Three-dimensional Structure of the Tryptophan

THEJOURNAL OF BIOLOGICAL CHEMISTRY

Vol. 263, Issue No. 33,

of November 25, pp. 17857-17871,1988 Printed in U.S.A.

Three-dimensional Structure of the Tryptophan Synthasea& Multienzyme Complexfrom Salmonella typhimurium* (Received for publication, June 20, 1988)

C. Craig HydeSg, S . Ashrafudin AhmedlIII, Eduardo A. PadlanS, Edith W.MileslI, and David R. DaviesS From the$Laboratory of Molecular Biology and the llLaboratory of Biochemical Pharmacology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892

The three-dimensionalstructure of the ad32 complex of tryptophan synthase from Salmonella typhimurium bas been determined by x-ray crystallography at 2.5 A resolution. The fourpolypeptide chains are arranged neady linearly in an aB@aorder forming a complex 160A long. The overallpolypeptide fold of the smaller a subunit, which cleaves indole glycerol phosphate, is that of an %fold a/B barrel. The a subunit active site has been located by difference Fourier analysisof the binding of indole propanol phosphate, a competitive inhibitor of the Q subunit and a close structural analog of the natural substrate. The larger pyridoxal phosphate-dependent B subunit contains two domains of nearly equal size, folded into similar helix/sheet/helix structures. The binding site for the coenzyme pyridoxal phosphate lies deep within the interfacebetween the two B subunit domains. The active sites of neighboring a and B subunits are separated by a distance of about 25 di. A tunnel witha diameter matching that of the intermediate substrate indole connects these active sites. The tunnel is believed to facilitate thediffusion of indole from its point of production in the a subunit active site to the siteof tryptophan synthesis in theB active site and thereby prevent escape its to the solvent during catalysis.

Tryptophan synthase (EC 4.2.1.20) catalyzes the final reaction in L-tryptophan biosynthesis. (For reviews, see Refs. 1-5, and for a historical account, see Ref. 6.) The bacterial enzyme is a bifunctional, bienzyme complex with an a& subunit composition (MI = 143,000) and thus is a simple model of multienzyme complexes. The a subunit (MI = 29,000) alone catalyzes the cleavage of indole 3-glycerol phosphate, termedhere the a reaction. The ,f3 subunit (Mr= 43,000) usually exists as a dimer, contains two molecules of the cofactor pyridoxal phosphate per dimer, and catalyzes the synthesis of L-tryptophan from indole and L-serine, termed the /3 reaction. When the a and p subunits combine to form the a& complex, the rates of the a and p reactions and the

* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. To whom correspondence should be addressed Laboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, Bldg. 2, Rm. 316, National Institutes of Health, Bethesda, MD 20892. 11 Present address: Dept. of Biochemistry, The Bowman Gray School of Medicine, 300 S. Hawthorne Rd., Winston-Salem, NC 27103.

affinities for substrates in thesereactions are increased by 12 orders of magnitude. The physiologically important reaction catalyzed by the a& complex, termed the a-preaction, is the sum of the a and p reactions. cy

reaction: Indole 3-glycerol-P + indole

@ reaction:

+ ~-glyceraldehyde-3-P

Indole + L-serine * L-tryptophan

a-(3reaction:

Indole 3-glycerol-P

+ L-serine * L-tryptophan

+ Hz0

+ ~-glyceraldehyde-3-P+ Hz0

In the overall a-p reaction, indole produced in the active site of the a subunit becomes a substrate for the active site of the p subunit where a pyridoxal phosphate-dependent /3 replacement reaction with L-serine yields L-tryptophan. There is evidence that indole does not appear as afree intermediate in solution during the course of this reaction (7-9). Various mechanisms have been proposed to account for these observations andwill bediscussed in light of the three-dimensional structure. Tryptophan synthase has been the subject of many important genetic and biochemical studies, and it is of interest to correlate the findings from thesestudies with thethreedimensional structure of the enzyme complex. The tryptophan synthase a& complex from Escherichia coli has been crystallized (lo), but crystals suitable for x-ray crystallography have not been obtained. In contrast,crystals of the enzyme from Salmonella typhimurium have been obtained by vapor diffusion from solutions of polyethylene glycol and spermine and yielded good preliminary x-ray data (11).The three-dimensional structure hasrecently been obtained at 2.8 A resolution (12) and is reported here at the current resolution of 2.5

A.

CRYSTALLOGRAPHIC STRUCTURE DETERMINATION

Crystals of the a& complex from S. typhimurium weregrown under conditions reported earlier (11) using enzyme prepared from the over-producing strain of S. typhimurium TB2211 containing the multicopy plasmid pSTH8. Both standard and recently improved purification procedures (5,13) were used.The “sandwich box” crystal growing technique (14) yielded the best crystals. Grown crystals were stabilized by addition of 20% (w/v) polyethylene glycol 8000 to the crystallization buffer. Pyridoxal phosphate at 0.6 mM was added to the stabilization buffer for 2 days prior to datacollection from native crystals. Location and Refinement of Heavy Atom-Heavy-atom derivatives for use in the multiple isomorphous replacement method were prepared by soaking native crystals for 1-7 days in 3.0 ml of a 20% polyethylene glycol 8000stabilizing solution in which the heavy-atom reagent had been either dissolved or suspended at concentrations from 0.1 to 3.0 mM. The useful derivatives included methylmercuric

17857

Structure of the Tryptophan Synthase Multienzyme

17858

acetate, merthiolate, 2,5-bis(chloromercuri)fran (15), and merthiolate with enzyme in which the sulfhydryls were previously blocked with three equivalents of iodoacetamide per a/@ subunit pair. Native and heavy-atom derivative diffraction data were collected using the MAXD area detector facility at the University of Virginia (16) and the Mark I1 area detector facility at theuniversity of California, San Diego (17). Data extending to about 2.2 A, but reasonably complete only to 2.5 A nominal resolution were collected from a native enzyme crystal. Diffraction data to 2.8 8, resolution were collected from four mercury derivatives which showed significant diffraction intensity changes in-15"precession photographs. The unitcell parameters were a = 184.5 A, b = 61.1 A, c = 67.7 A, @ = 94.7" (space group C2) in the presence of the 20% polyethylene glycol 8000 stabilizing solution, differing slightly from earlier reported values (11). Heavy-atom positions from the four derivatives found by difference Patterson analysis are summarized in Table I. Positions and occupancies from all four derivatives were simultaneously refined using a program originally written by Michael Rossmann and modified by Ten Eyck and Arnone (18). All of the derivatives showed multiple substitution by binding with differing selectivities to some or all of the 8 cysteine residues in each a/@subunit pair. Multiple isomorphous replacement phase information from the four derivatives were combined, resulting in a mean figure-of-merit of 0.48for 17,866reflections

TABLEI Refined heavy-atom parameters Refined heavy-atom positions and occupancies. All mercury binding sites are near the eight cysteine sulfhydryls in the asymmetric unit (an a/@ subunit pair). Abbreviations: s, soaking time, in days; c, concentration of reagent; n, number of unique structure factor amplitude measurements; r, nominal resolution; p . ~ . ,phasing power of the derivative, expressed as theratio of the root-mean-square heavyatom structure factor amplitude to the root-mean-square lack-ofclosure error; Occ., refined occupancy on an arbitrary scale; X, Y, Z, fractional coordinates; B, temperature factor, in units of A'. Methylmercuric acetate s = 2; c = 1 m M ;n = 14,893; r = 2.8

Site

CYS-154~~ CYS-1308 CYS-620 Cys-118a Cys-l70@ CYS-3406 Cys-8la CYS-2308

A;pp. = 1.57

occ.

X

Y

z

B

2.53 1.64 2.40 2.46 1.58 1.17 1.86 1.18

0.2382 0.3910 0.4464 0.2964 0.3840 0.4446 0.1925 0.4747

0.2497 0.2687 0.1039 0.4421 0.3088 0.2727 0.0078 0.2597

0.1491 0.9103 0.8189 0.4711 0.1877 0.8892 0.5616 0.2706

28.9 29.1 28.7 26.6 33.8 29.8 32.7 40.2

Merthiolate

Site

CYS-154a CYS-1306 CYS-628 CYS-118~~ CYS-1708 CYS-3408 Cys-8la

occ.

X

Y

z

B

2.37 1.14 1.96 2.35 1.63 1.13 1.39

0.2382 0.3899 0.4465 0.2973 0.3849 0.4442 0.1883

0.2538 0.2733 0.1076 0.4430 0.3114 0.2734 0.0286

0.1478 0.9093 0.8194 0.4745 0.1863 0.8888 0.5700

27.8 28.7 20.4 28.7 26.2 25.0 24.8

occ.

A;

Site

occ.

X

Y

Z

B

CYS-15401 CYS-628 Cys-118a Cys-170@ Cys-8la

1.72 0.76 1.54 0.81 1.22

0.2382 0.4462 0.2971 0.3847 0.1885

0.2500 0.1049 0.4386 0.3067 0.0250

0.1482 0.8181 0.4750 0.1857 0.5712

34.5 16.0 22.4 14.6 17.1

E = 7;

2,5-Bis(chloromercuri)furan c = 0.1 m M (saturated solution); n = 9,426; r = 2.5 p.p. = 1.02

Site

Cys-62@ Cys-118a Cy~-170@ Cys-8la

0.65 1.23 0.89 1.15

X

Y

Z

0.4445 0.2959 0.3854 0.1895

0.1032 0.4382 0.3075 0.0206

0.8224 0.4692 0.1877 0.5617

%

1 2

3

Merthiolate-iodoacetamide 2.8 p.p. = 1.26

s = 2; c = 0.5 mM;n = 14,589; r =

extending from 15 to2.8 8, resolution and were used to construct an electron density map. This initial map revealed the outline of the protein-containing regions of the unit cell, but density features were poorly connected and the course of the polypeptide chain could not be traced. The clarity of the multiple isomorphous replacement map was improved using the ISIR/SAS package of programs by B. C. Wang (19) which combined the heavy-atom phase information with phases computed from a modified, solvent-leveled electron density map. Starting phase information consisted of the multiple isomorphous replacement phases based on the four mercury derivatives to 2.8 A resolution as well as anomalous scattering data from the iodoacetamide-blocked merthiolate derivative to 3.2 A resolution. The estimated solvent fraction of 50% for tryptophan synthase crystals was not imposed until the final cycle after having been set at35 and 45% in two previous cycles. No phase or amplitude extension was performed. On completion of the final cycle, the mean figure-of-merit increased from a starting value of 0.48 to 0.76 with an accumulated mean phase shift of 38.2" from the initial phasesfor the 17,786unique reflections. The course of the image enhancement procedure is outlined in Table 11. The resulting improved electron density map was sufficiently clear to allow chain tracing and model fitting to proceed. A portion of this electron density map is shown in Fig. L4. Atomic Modeling-It was possible to construct an atomic model containing nearly 80% of the 665 amino acid residues in-the a/@ subunit pair (the asymmetric unit) from this improved 2.8 A resolution electron density map. The amino acid sequences used in modeling the electron density maps were from the S. typhimurium a (20) and 8 subunits (21) deduced from the gene sequences. Modeling was performed on an Evans and Sutherland PS330 graphics system using the Rice University version of the FRODO software (22). The initial atomic model comprising 80% of the structure was subjected to restrained least-squares refinement using an array-processor version (23) of the program PROLSQ by Hendrickson and Konnert (24, 25). About 100 cycles of refinement were interspersed with five cycles of manual rebuilding using computer graphics. During the first rebuilding cycle, where the model phase information alone was inadequate to define missing portions of the model, maps were constructed by combining solvent-leveled multiple isomorphous replacement phase information with phases calculated from the partial structure (26). Further refinement allowed exclusive use of calculated phase information from the refined partial model in constructing "fragment" 2F0-F,maps or OMIT maps (27). An example of the quality of the current OMIT maps is shown in Fig. 1B. The current atomic model consisting of 4,780 atoms (96% of the total) with individual temperature factors has been refined against 22,832 structure factor amplitudes (where IF1>3a(lF I )) extending to about 2.5 A resolution to a

TABLE I1 Electron density image enhancement by iterative solvent leveling

s = 1; c = 3 mM; n = 15,413; r = 2.8 A; p.p. = 1.67 -

Complex

A; B 25.4 30.3 33.0 35.9

35 45

50

7%

43.2 1 22.3" 0.64 1 29.5 0.70 28.92 32.5 0.73 3 0.7526.8 25.74 35.5 0.75 0.75 1 2 0.75 25.93 37.7 0.76 25.4 4 38.2 0.76

33.5 34.3 36.5 37.1

27.2 26.4

"The filter number refers to the different solvent masks corresponding to different imposed solvent contents. *Solvent content fraction enteredinto the automated protein envelope program ENVELP of the ISIR/ISAS package and used to level that fraction of the unit cell volume having the lowest electron density values. Mean figure-of-merit at end of the cycle for 17,786unique reflections. Initial mean figure-of-merit from multiple isomorphous replacement data was 0.48. Mean accumulated phase shift from the initial multiple isomorphous replacement phases. 1 FobI where F,,& are the observed ' R = I Fobs - Fcdc 1 are calculated by Fourier inverstructure factor amplitudes and FCalc sion of the modified electron density map.

/x

Structure of the Tryptophan Synthase MultienzymeComplex

17859

FIG. 1. A, current model superimposed on the original 2.8 A multiple isomorphous replacement electron density map improved by iterative solvent leveling. The portion of the map shown is located in the middle of the @ subunit Cdomain. B, model as shown in A , but superimposed on the current OMITMAP electron density map at 2.5 A resolution. C, positive difference density features, contoured at 2.0 times the rootmean-square density level of the map, showing the presence of the bound substrate analog, indole propanol phosphate, at the CY subunit active site. The indole, propyl, and phosphate moieties of the inhibitor are clearly indicated by the positive difference densities. Strong features adjacentto thephosphate group suggest that residues 234 and 235 move from left to right by over 1 A when the substrate binds. Atoms C3 and C3' define the bond that is cleaved by the enzyme in the natural substrate, indole glycerol phosphate.

current R-factor of 25.5%. The root-mean;square deviation from ideality in bond lengths and angles is 0.01 A and 1.3",respectively. Preliminary coordinates willbe submitted for depositions in the Protein Data Bank (28) to coincide with this publication. Currently,

all but the following residues have been fitted: 56-58, 178-191, and 266-268 in the CY subunit and residues 1-8 and 394-397 in the @ subunit (the extreme N and C termini). Localizing the Active Site of the LY Subunit-In order to identify the

Structure of the Tryptophan Synthase MultienzymeComplex

17860

active site region of the a subunit, diffraction data to 2.5 A were collected from a native enzyme crystal which had been soaked for 4 days in a 20% polyethylene glycol 8000 stabilizing solution containing 0.4 mM indole propanol phosphate. This compound is a stable competitive inhibitor of the a subunit and a close structural analog to the natural substrate, indole glycerol phosphate (29). The binding of indole propanol phosphate was analyzed using a difference Fourier map at 2.5 A resolution computed using phases derived from the native enzyme model. The positive difference density features of this map in the active site region of the a subunit are shown in Fig. 1C.

Fig. 2, A and B. The two tightly associated /3 subunits are at the center of the complex whilethe two a subunits are located distant from eachother at opposite ends of the 0 2 dimer. This finding is consistent with previous results obtained by x-ray scattering (30)and neutron scattering (31).The overall length of the tetramer is about 150 A. The active site of each a subunit is located near the interface with the /3 subunit, whereas the active site of each /3 subunit is deeply buriednear the center of the /3 subunit (Fig. 2, A and B). The two active sites of neighboring a and b subunits are separated by a RESULTSAND DISCUSSION distance of about 25-30 A. The a subunit, which has an 8Overall Structure of the Complex-The quaternary structure fold a//3 barrel fold (see below), is "tilted" relative to the /3 of the tetrameric complex has an extended, nearlylinear a&3a subunit such that the central axis of the a subunit barrel subunit arrangement; two views of this complex are shown in forms an approximate 20" angle with a l i e drawn between

a

FIG. 2. A, viewof the S. typhirnuriurn OC& tryptophan synthase complex looking approximately down the 2fold axis of symmetry between cro subunit pairs. The smaller 01 subunits (blue) are distant from each other on opposite ends of the p subunit dimer. The @ subunit N-terminal residues (1-204) and C-terminal residues (205397) are shown in yellow and red, respectively. The dot surfaces highlight the positions of bound indole propanol phosphate ( r e d ) in the active sites of the a subunits and the coenzyme pyridoxal phosphate (dark blue) in the active sites of the fi subunits. A tunnel which connects the two active sites (light blue) is shown in one LYPsubunit pair. B, view of the multienzyme complex with the 2-fold axis vertical.

Structure of the Tryptophan Synthase Multienzyme the two active sites (Fig. 2B). Fig. 2 also shows a tunnel extending between the a and p active sites whichwillbe discussed below. Since the active sites of the two. subunits in the a282 complex are quite distant (over 25 A) it seems unlikely that residues from one p subunit contribute to the active site of the other ,8 subunit. As a consequence, the a/P subunit pair is the functional unit. A smoothed backbone representation of an a l p subunit pair is shown later in Fig. 8A. The two a / @ subunit pairs in the tetramer are, in a sense, back-to-back with each other. Thus theformation of the a2/32tetramer does not appear necessary for catalysis but may play a role in stabilizing the conformations of the P subunits. The two P subunits interactover a broad, nearly flat surface through which a dyad axis of symmetry passes (Fig. 2 A ) . Part of the N-domain of each p subunit interacts with part of the C-domain of the complementary P subunit. A variety of forces, including van der Waals, hydrophobic, hydrogen bonding, and ion pair interactions all appearto contribute to thestabilization of the contact surfac?. This buried surface is estimated to have an area of 1,440 A2 using the program MS (32) with standard van der Waals radii (33) and a probe sphere radius of 1.7 A. Studies of the pressure dissociation of the holo p 2 dimer demonstrate that thep subunits are tightlyassociated; the dissociation constant at 1bar is K d = 3.7 X 10”’ M (34). (The p/p subunit contact surface can best be seen in Fig. 8, B and C , described below.) The interface between the a and ,8 subuni!s is mostly hydrophobic in character. Apprpximately 1190 A* of surface area of the a subunit and 1110 A2 of the p subunit are buried at thea / @interface. For comparison, two recently determined lysozyme. anti-lysozyFe Fab structures have reported contact surface areas of 750 A2 (35) and 690 k (36). Thus the areas of interaction between a and (3 subunits and between the two P subunits are more extensive than the contact surfaces in these antibody-antigen complexes. Structure of the a Subunit of Tryptophan Synthase-The tertiary fold of the a subunit follows the 8-fold a//? barrel motif first observed in triosephosphate isomerase (37) and now found in twelve other enzymes (summarized in Ref. 38). Interestingly, this includes both domains of the bifunctional enzyme phosphoribosylanthranilate isomerase:indoleglycerolphosphate synthase from E. coli (39, 40), which catalyzes the two reactions in the tryptophan biosynthetic pathway immediately preceding the tryptophan synthasereaction. The a l p barrelstructure, shown in Fig.3, can best be described in termsof a “canonical” form in which the molecule is built up from eight repeating supersecondary structural units each comprised of a P-strand followed by an a-helix. The successive strands pack adjacent to each other to form a strongly twisted, extended parallel sheet. The twist is such that thesheet turns on itself to bring the eighth strand around to pair with the first, therebygiving the appearance of a barrel with the strands asstaves. The middle of the barrel is packed mostly with hydrophobic residues. The eight helices, connected to both ends of the strands by loops, pack closely in parallel around the periphery of the barrel. It should be noted that the strands and helices are tilted by about 35” relative to the central axispassing through the roughly cylindrical structure. A previous comparison of the three-dimensional structures of triosephosphate isomerase, pyruvate kinase, and 2keto-3-deoxy-6-phosphogluconatealdolase (42, 43), all of which possess one a l p barrel, showed that the eight strands and outer helices are nearly superimposable in the differen: structures with a root-mean-square difference of about 3.1 A between about 150 Ca positions (43). An alignment of triosephosphate isomerase with the a subunit of tryptophan syn-

Complex

17861

FIG. 3. Schematic view of the a subunit of tryptophan synthase. Strands are shown as flattened arrows with arrowheads at their C termini. Helices are represented as cylinders and are labeled on their N termini. The 01 subunit numbering is made in reference to a simpler canonical a/@barrel with eight strands and eight peripheral helices. See Table I11 for secondary structure assignments. Additional helices 2’ and 8’ are located above the active site and helix 0 is located below on the opposite side of the subunit. The active site is indicated by the position of the bound inhibitor, indole propanol phosphate, shown in a ball-and-stick representation. The light stippling in the loops following strands 2 and 6 shows the portions of the structure that are ill defined at present. The loops following strands 2, 3, 4,and 5 in the 01 subunit make important contacts with the @ subunit. The P in one of these loops indicates the site susceptible to cleavage by trypsin (Arg-188) which delineates the N- and C-terminal fragments. From this point of view, the @ subunit would block the view of roughly the upper half of the figure, i.e. helices and strands 1-5. The phosphorous and indole ring nitrogen atoms are shaded. Production of the schematic drawingswas aided by a computer graphics program (41).

thase by an automated least-squares procedure using a basis set of 156 Ca atom pairs currently give? a root-mean-square deviation between paired atoms of 2.9 A. This high level of structural homology between triosephosphate isomerase and the tryptophan synthase 01 subunit is interesting because the two enzymes share a reactant:one product of the tryptophan synthase a reaction, glyceraldehyde 3-phosphate, is also a substrate of triosephosphate isomerase. This suggests that the structural motif common to these two catalytic units may have been preserved for functional reasons. The various members of the a / @barrel class are distinguished from each other in amino acid sequence, in the lengths of the well defined secondary structural elements, and in the length and conformations of the connecting loops between helices and strands. The assignment of secondary structure to the amino acid sequence is given in Table 111. The helices and strands corresponding to canonical a/P barrel elements have been numbered consecutively 1-8. Inthe a subunit of tryptophan synthase, an extra helical segment designated helix “0” precedes the first strand and acts to capbottom the of the barrel, i.e. the side of the subunit opposite the active site. This is unlike the indoleglycerol-3-phosphate synthase domain of phosphoribosylanthranilate isomerase:indoleglycerolphosphate synthase in which an extra N-terminal helix is located at theactive site (40). Another prominent deviation from the canonical form is an insertion of 26 residues, 53-78 between strand 2 and helix 2 which is one of the most highly conserved regions of the a subunits among 10 microbial species (44). Some residues, 55-58 located at the interface of the a and p subunits, have poor electron density featuresandappear

17862

Structure of the Tryptophan Synthase MultienzymeComplex TABLE I11 Secondary structure assignments Assignments of secondary structure were made by visual inspection of the model.H,Symbols: a-helices;B, strands in P-sheets; t, residues at the center of r,sharp all other turns;structural classes including “random coil” ell defined at present and “missing” from the The current amino acid model. conformations;?, for regions wnot sequences are from Refs. 20 and 21. Designations of helices and strands shown arebelow each line. Ina the subunit, helices 0, 2’,and 8’are “noncanonical” with respect a simple to 8-fold a/B barrel structure and have therefore been given special designations. a subunit”

50

40

1 30 0

20

60

MERYENLFAQLNDRREGAFVPFVTLGDPGIEQSLKIIDTLIDAGADALELGVPFSDPLAD

HHHHHHHHHHHHttBBBBBBBBBBttttrrttHHHHHHHHttrrBBBBBBrrr???rr Helix 0 Strand 1 Helix 1 Strand 2 7090

80

100

120

110

GPTIQNANLRAFAAGVTPAQCFEMLALIREKHPTIPIGLLMYANLVFNNGIDAFYARCEQ rrHHHHHHHHHHHrrrrrHttrrrBBBBBBBBttttrttr-

Helix Helix 2’ 180

170

160

150

140

Strand 3

2

Helix 3

130

VGVDSVLVADVPVEESAPFRQAALRHNIAPIFICPPNADDDLLRQVASYGRGYTYLLSRS

rrrBBBBBttrrrrrrrHHHHHHHHrrBBBBBBBrrrrrHHHHHHHHHrrrrBBBBB??? Strand 4 Helix 4 Strand 5 Helix 5 Strand 6 240

230

220

210

200 190 GVTGAENRGALPLHHLIEKLKEYHAAPALQGFGISSPEQVSAAVRAGAAGAISGSAIVKI ???????????HHHHHHHHHHHrrrrrBBBBBrrrrHHHHHHHHHrrrBBBBttHHHHHH Helix 6 Strand 7 Helix 7 Strand 8 Helix 260 250 IEKNLASPKQMLAELRSFVSAMKAASRA HHttttr ?? Helix8’ 8 B subunit” ~

60

50

40 1 30 0 20 MTTLLNPYFGEFGGMYVPQILMPALNQLEEAFVRAQKDPEFQAQFADLLKNYAGRPTALT ????????rrrrrrrrrrrttrrHHHHHHHHHHHHHHrrHrrrrrrBBB Helix 1 Helix 2 Strand 70 80 90 100 110 120 KCQNITAGTRTTLYLKREDLLHGGAHKTNQVLGQALLAKRSA BBrrttttrrBBBBBBBrrrrttrrrHHHHHHHHHHHrrrrrrBBBBBBBrrrrm

Strand 2

1

170

160

150

Helix 3

Strand 3

140 130 180 LASALLGLKCRIYMGAKDVERQSPNVFRMRLMGAEVIPVHSGSATLKDACNEALRDWSGS HHHHttrBBBBBBBrtttrrttrrHHHHHHttrBBBBBBrrrrrr-ttr 4 Strand 4 Helix 5 Strand 5

240

230

220

210

Helix

Helix 6

200 190 YETAHYMLGTAAGPHPYPTIVREFQRMIGEETKAQILDKEGRLPDAVIACV~SNAIGM ttrBBBBBrrrrrrrrrHHHHHHtt-rrrrBBBBBBBrrrm Strand 6 Helix 7 Helix 8

Strand 7

Helix

300

290

280

270

260 250 FADFINDTSVGLIGVEPGGHGIETGEHGAPLKHGRVGIYFG~P~T~IEESYSI HHHHrrrrrBBBBBBBrrttrrrrrrrrrrttrrrrrrrttrrrrrrrrttrrrrrrrrr 9 Strand 8

360

350

340

330

320 310 SAGLDFPSVGPQHAYLNSIGRADYVSITDDEALEAFKTLCRHEGIIPALESSHALAHALK rrrrrrrrrrHHHHHHHHHrrBBBBBBr-rrrrrrrHelix 10 Strand 9 Helix 11 Helix 12 390

380

370

MMREQPEKEQLLWNLSGRGDKDIF”VHDILKARGE1

HHHHrrrrBBBBBBBrrrrrrrHHHHHHHHHHH???? Strand 10 Helix 13 a

but

Note: Residues 103-106 of the a subunit have not been designated as

and 136-138 of the P subunit both having helical conformations

highly mobile despite being somewhat buried. This insertion also contains Asp-60 which may have a catalytic role (see below). Helix 2‘ (residues 63-73) comprises the remainder of

form a distorted one-turn helix because of their short

length.

this 26-residue inserted region. An additional 2.5-turn ahelical segment (helix 8’) is present in the loop connecting strand 8 to helix 8 and oriented so that the N-terminal end

Structure of the Tryptophan Synthase Multienzyme Complex

FIG. 4. A, stereo view of the active site region of the a subunit looking approximately downthe central axis of the 8-fold a/@ barrel and from a direction fromwhich the substrate might enter. The position and conformation of the bound inhibitor indole propanol phosphate is shown in yellow at the center. The surrounding amino acid residues of the a subunit are shown in their conformations in the native, unligated formof the enzyme. Glu-49, Asp-60, and Tyr-175 have been highlightedin pink because oftheir suspected catalytic or special substrate binding roles. The remaining residues highlighted in white are either close to or makedirect contact with the inhibitor and may therefore have rolesin substrate binding and in conferring specificity.B, locations of missense mutations in the a subunit active site.The position of the bound competitiveinhibitor indole propanol phosphate is shown near in surfaces, the center of the figure in a blue van derWaal's dot surface.The remaining nearby residues showndot including Phe-22, Glu-49, "175, Gly-211, Gly-213, Gly-234, and Ser-235, are all previously identified sites of mutations. These residues are clustered in the active site and either near or withindirect contact with the inhibitor. The residues are shown in their conformationsin the native, unligatedform of the enzyme. C, pyridoxal phosphate binding site at the interface of N-domain (yellow) and C-domains (red) of the 0 subunit. Residues that may be important in binding the coenzyme have been labeledand highlighted (see text). The Schiff base complex ofLys87 and pyridoxal phosphate is shown highlightedin white, the C=N double bond betweenthem in yellow. P, N l , and 03 designate the phosphorous atom, pyridoxal ring nitrogen, and hydroxyl oxygen atoms of the coenzyme, respectively; the Schiff base nitrogenis labeled NZ.The putative binding site for the substrate L-serine, deduced from modeling studies,is indicated by a small sphere (orange dot surfme).

17863

17864

Structure of the Tryptophan Synthase Multienzyme Complex

of this short helix points toward the active site region. The a Subunit Active Site-The active center of the a subunit positive end of the helix dipole could play a role in binding has been located by binding the competitive inhibitor indole the phosphate group of indole glycerol phosphate (see below). propanol phosphate (29) to the crystalline enzyme. A differResidues 179-191 between strand 6 and helix 6, appear to ence electron density map at 2.5 A resolution shows strong be highly disordered in the native enzyme crystal; much of it positive features clearly outlining and delineating the indole is invisible in the electron density map. Although this loop ring and propyl and phosphate groups of the inhibitor (Fig. contains a site (Arg-188) which is susceptible to proteolysis IC). Strong positive and negative difference density features by trypsin in the a& complex (45, 46), proteolysis is not in theneighboring protein atomssuggest local conformational responsible for the weak electron density since a sodium adjustments that apparently accompany substrate binding. A dodecyl sulfate-polyacrylamide gel of enzyme crystals showed more complete modeling and refinement of this enzymeonly twoprominent bandscorresponding in size to the stand- inhibitor complex is nowunderway. ard a and @ subunits. Furthermore, replacement of another Indole propanol phosphate binds in a predominantly hyresidue in this loop,Arg-179,by site-directed mutagenesis drophobic “pit” formed just below the surface of the subunit demonstrates that this residue is not essential for catalysis at the top of the central barrel. Similar active site locations although it may play a role in the transmission of ligand- have been identified in several other a / @barrel enzymes, dependent effects from the a subunit to the @ subunit (47). including triosephosphate isomerase (37, 43, 60) and phosAlthough the remaining loops between helices and strands phoribosylanthranilate isomerase:indoleglycerolphosphate are unremarkable with respect to size or conformation, two synthase (39,40). The conformation and position of indole propanol phosof them, the stretches between strands 3 and 4 andhelices 3 and 4, form much of the contact surface with the 0 subunit. phate bound in the a subunit active site is shown in Fig. 4A. Position 109, also in this region, was previously identified by The active site is formed from the amino acid side chains of chemical modification studies to be in the site of interaction residues near the C-terminal endsof the eight central strands. The inhibitor appearsto contact side chain atoms or backbone between the E. coli a and @ subunits (48). Domain Structure of the a Subunit-While the %fold a / @ atoms from all eight strands or from the loops immediately barrel proteins seem to consist of a single structural domain following them. Most of the amino acids that are highly (42),there is considerable evidence that the a subunit of conserved in the sequences of the a subunit from 10 microtryptophan synthase containstwo folding domains. The dem- organisms (44) are clustered in this region. The binding site onstration of complementation between mutant a subunits in for the indole moiety of the substratecontains several hydroartificial dimers formed following exposure to 6 M urea first phobic residues, including: Phe-22, Leu-100, Tyr-102, Leusuggested an interaction between two domains (49, 50). Lim- 127, Ala-129, Ile-153, and Tyr-175. Ile-64 and Leu-177 are ited proteolysis of the a subunit at Arg-188 yields two frag- neighboring hydrophobic residues but probably do not make ments which can independently refold either fully (residues direct contact with the inhibitor. The propylphosphate por1-188) or partially (residues 189-268) (45, 46,51, 52). The tion of the inhibitor extends at approximately a right angle stepwise unfolding of the a subunit induced by guanidine to theindole ring with the phosphate group binding between hydrochloride (53-55) or by urea (56,57)parallels the unfold- the peptide loops containing residues 211-213 and 234-235. ing of the two proteolytic fragments which correspond to the A surprising finding is that no basic amino acid side chain two domains (51,52). An examination of the crystal structure binds the phosphate. It appears instead that hydrogen bonds of the a subunit in the az@zcomplex shows that Arg-188 (the with backbone atoms and the side chain hydroxyl of Ser-235 site of proteolysis) is in a highly mobile surface loop which are primarily responsible for binding, rather than ion pair connects strand 6 and helix 6. Cleavage at Arg-188 would interactions. Since the N terminus of a short helix (helix 8’) result in an N-terminal fragment containing the first five near Ser-235 points toward the phosphate binding site, helixhelix/strand structural units and strand 6 (a5/Ps),and a C- dipole interactions also may be involved. From the current terminal fragment, containing helix 6 and the last two of difference electron density map, it appears that the indole binding site is “preformed” in the native enzyme; few confortheseunits (a3/@Z). These folding studies (45, 46,51,52) combined with the crystallographic results lead to theconclu- mational changes are indicated around the indole-binding sions that the N-terminal part of the a / @barrel can fold residues. On the other hand, binding of the phosphate seems independently and that partial a/@barrels are much more to induce large conformational and positional changes in the stable than would have been expected (58). Small-angle neu- nearby residues, Gly-234 and Ser-235 in particular (Fig. IC). tron scatteringdata (31) and small-angle x-ray scattering data Two residues in the a subunit active site, Glu-49 and Asp60, may be catalytic residues. Glu-49 is found deep inside the (30) are compatible with a compact form of the a subunit. While this work on tryptophan synthase was in progress, hydrophobic core of the protein with its carboxylate located two independent groups have predicted the structure of the a near the scissile bond and the presumed location of the 3’subunit (44,59)by comparing secondary structure predictions hydroxyl of the natural substrate. Recent studies with 19 from the sequences of several microbial species. In one study mutants at position 49 indicate that Glu-49 is essential for (44), an 8-fold a/@barrel structure was predicted. Although activity and strongly suggest that it is a catalytic residue (61, the predicted assignments of major secondary elements to the 62). Our results supportconclusions from NMR studies about amino acid sequence showed good agreement with the results the spatial relationships between Glu-49 and tyrosyl residues of x-ray crystallography reported here, even better agreement in the a subunit (63). The other putative catalytic residue in has been subsequently achieved by an automated prediction this region, Asp-60, is highly conserved. The carboxylate of procedure.‘ In the second study (59), a @-sheetla-helix struc- Asp-60 seems to pair with the indole nitrogen atom of the ture was predicted initially but was re-evaluated and found to inhibitor. Substitution of this residue by Asn (64) or by Asn, be consistent with an a / @barrel structure after the prelimi- Ala, or Tyr3 results in loss of a subunit activity. Although the hydroxyl of Tyr-175 is also found close to the substrate and nary crystallographic results became available.2 near the scissile bond, the recent finding that substitution of T. Nierman and K. Kirschner, unpublished results. We shall provide a detailed evaluation of these predictions in a later publication.

S. Nagata and E. W. Miles, unpublished results.

Structure of the Tryptophan Synthase MultienzymeComplex Tyr-175 by phenylalanine does not result in loss of catalytic activity3 indicates that thehydroxyl of Tyr-175 does not play an essential role. Residues 22,49, 175, 177, 211, 234,and 235, which are the sites of missense mutations which inactivate the LY subunit from E. coli, and residue 213, which is changed in some second site revertants (1,65), are located all near the bound inhibitor (Fig. 4B).The structure of the active site also confirms the suggestion based on studies of spcond site revertants that residues 175, 177, 211,and 213 are in close spatial proximity (66). Structure of the P Subunit-The alignment of secondary structure with the amino acid sequence of the P subunit is given in Table 111. The P subunit contains two domains of nearly equal size separated by a plane approximately parallel to thedyad axis that relates the two P subunits inthe complex. These two domains can be clearly seen in Fig. 2, A and B, where they are colored in yellow and red. The active site which contains the binding site for the coenzyme pyridoxal phosphate is formed deep within the subunit between these two domains. Since these domains are largely derived from the N-terminal sequence (residues 1-204) and the C-terminal sequence (residues 205-397), we will refer to them as the Ndomain and C-domain, respectively. One stretch of the Nterminal sequence (residues 53-85) “crosses over” into theCdomain, forming the first two strands of a @-sheet at the center of the domain, so these residues have been designated C-domain residues. The structure of the N-domain of the P subunit is shown schematically in Fig. 5. The core of the N-domain, residues 85-190, is formed from four strands, designated 3-6 in Fig. 5 , with three helices (helices 3, 4, and 5 ) packed on one side of the sheet, and a fourth helix (helix 6) packed on the other. The strands are aligned adjacent to each other in parallel in

ETA SUBUNIT

from C-domain

FIG. 5. Schematic view of the foldingpattern and secondary structural elements of the fi subunit N-domain viewed from domain would the direction of the C-domain; nearly the entire be hidden from view by the fi subunit C-domain. See Table I11 for secondary structure assignments. The core of this domain is formed by a four-strandparallel &sheet (strands 5 4 . 3 , and 6) packed on both sides by four helices. The N-terminal helices 1 and 2 wrap around the globular core of the domain. Residues 53-85 cross-over and are closely associated with the C-domain. The pyridoxal phosphate. Lys-87 Schiff base complex is shown in a ball-and-stickmodel, with the phosphate group located toward the left. The phosphorous and pyridoxal ring nitrogen atoms are shaded.

17865

fcl BETA SUBUNIT C-DOMAIN

from N-domain

FIG. 6. Schematic view of the folding pattern of the fi subunit C-domain viewed from the N-domain. The C-domain is made of a six-strand &sheet surrounded by helices. Residues 53-85 forming strands 1 and 2 are from the N-terminal portion of the subunit. P shows the site susceptible to proteolytic cleavage at Lys272, Arg-275, and Lys-283, which generates the F1 andF2 fragments. This cleavage site is within a stretch of residues (260-310) which apparently do not have well defined secondary structure. The pyridoxal phosphate. Lys-87 Schiff base complex is shown in a ball-andstick model with the phosphate group located toward the lower right.

the order 6, 3, 4, and 5 with a familiar overall left-handed twist. The four sets of alternating helix and strands (typical CY/@ tertiary structure (42)) arearranged such that thefourth helix (helix 6) extends across the back side of the sheet and allows the fourthstrand(strand 6) to pair with the first (strand 3). Thisthree-tiered helix/sheet/helix sandwich, forming the core of the N-domain, is wrapped loosely by two helices arranged nearly at right angles to each other (helices 1and 2 from the N-terminal region of the molecule). The structure of the C-domain of the P subunit is shown schematically in Fig. 6. The long helical segment connecting the two domains appears to be “kinked” at position 204-205, the major dividing point between domains, and has therefore been designated as two helices, helix 7 and8. The core of the C-domain contains a central@-sheetsandwiched between two layers of parallel-packed helices, much like the core of the Nterminal domain. The @-sheetcontains five parallel strands, 2,10, 7, 8, and 9in Fig. 6, and one anti-parallel strand, 1. The sheet is strongly twisted so that the sixth strand is nearly perpendicular to thefirst. The first of the five parallel strands, strand 2, contains residues 71-77 from the N-terminal part of the @ subunit. Three or four residues, 58-61, may prove to hydrogen bond to strand2 and have tentatively been assigned to strand 1 of the P-sheet, which runs anti-parallel to the other five strands. The more interior side of the C-domain contains three helices (helices 8, 9, and 10 in Fig. 6) which are packed parallel to each other, but which are anti-parallel to the strands. The center helix (helix 9) points directly away from the bound pyridoxal phosphate with itsNterminus directed toward the phosphate. On the opposite, more exterior, side of the C-domain’s P-sheet, only one prominent helix (helix 12) is found packed next to the sheet. This leaves strands 8 and 9 of the central sheet more exposed to solvent. Helix 11is packed nearly perpendicular to helix 12 with most of its surface exposed to solvent.

Structure of the Tryptophan Synthase Multienzyme

17866

The remaining residues of the C-domain form two features that are not part of the domain’s helix/sheet/helix sandwich structure. One is a 50-residue stretch containingresidues 260310 that folds in a complicated way and apparently lacks any well defined secondary structural elements. At several points, the polypeptide chain folds back on itself in anti-parallel fashion to form extended, hairpin loops. This region makes several contacts with the a subunit and contributes several residues to thewall that lines the substrate tunnel(see below). The other feature is a three-turn helix (helix 13 in Fig. 6) at the C terminus of the polypeptide chain that protrudes away from the center of the p subunit and into the solvent, an unusual configuration for globular proteins. The residues in helix 13are highly variable in p subunits from different organisms (67). Studies of the effects of limited proteolysis on the Pz dimer of tryptophan synthase have demonstrated that each ,8 protomer is composed of two independently folding regions or domains (68-71). Threesites of tryptic cleavage, Lys-272, Arg-275, and Lys-283 (see arrow, Fig. 6), have been identified in the “hinge region” between the two domains (68). The larger N-terminal proteolytic fragment, termed F1, and the smaller C-terminal fragment, termed F2, refoldindependently after denaturation and separation. The refolded fragments fail to bind ligands (pyridoxal phosphate, L-serine, or Ltryptophan) when separate, but regain this ability when reassociated. These observations led to an early suggestion that the binding site for the pyridoxal phosphate coenzyme and the active site of the p subunit might lie at the interface between the F1 andF2 domains (71). Studies of the @ d’lmer and of its proteolytic fragments during unfolding (72) and refolding (73) provide evidence that the F1 and F2 fragments correspond tostructuralintermediates in the pathway of folding and unfolding. However, these F1 and F2 folding domains are notidentical with the N-terminal and C-terminal structural domains of the S. typhimurium /3 subunit. The “hinge” between the F1 and F2 domains is located near the edge of the interface of the a and subunits on a side of the @ subunit opposite the dividing point between the N- and Cdomains (residues 204-205). The location of this hinge region near the cup subunit interface was previously predicted from studies which demonstrated that therate of proteolysis of the

Complex

pZ dimer was greatly reduced in the

a& complex (45) and from studies which showed that the cleaved p2 dimer cannot form a complex with the a subunit (70). Structural Homology between the N - and C-terminal Domains-Examination of the schematic figures of the folding patterns of the two @ subunit domains (Figs. 5 and 6)reveals that portions of both domains have similar folding topologies. The core of the N-domain contains four parallel strands with three helices packed on the interior side of the sheet, and a fourth helix packed on its exterior side. This same pattern is also found at thecenter of the C-domain. Thus, thefollowing secondary structural elements appear topologically equivalent: helices 3, 4, 5, and 6 (of the N-domain) and helices 8, 9, 10, and 12 (of the C-domain), respectively; and strands 3, 4, 5, and 6 (of the N-domain) and strands 7,8,9, and10 (of the C-domain), respectively. The cores of the two domains also possess a high level of structural homology, despite a lack of amino acid sequence homology, and are nearly superimposable. The Ca positions of both @ subunit domains were superimposed using the program ALIGN (written by G. H. Cohen using the algorithm of Needleman and Wunsch (74)), the result of which is shown in Fig. 7. Nearly half (about 90-95) of the residues in each domain appear to have conformations similar to those in the $her domain. For 73 Ca pairs which devicte by less than 4.0 A, the root-mean-square deviation was 2.2 A. Only eight amino acid identities are found between these 73 structurally equivalent Ca pairs (Fig. 7). The structurally homologous regions of the two domains are related by an approximate pseudodyad axis with a small translational component. To superimpose the C-domain on the N-domain, the C-domai! must be rotated 159” about an axis and then translated 5.5 A. The rotation axis is located near the phosphate of the coenzyme bound at the interface between the N- and C-domains (see below), and is tilted about 18”relative to the true dyad axis that relates the two a l p subunit pairs in the C Y Z ~ Zcomplex. The plane of the aromatic ring of the pyridoxal phosphate is roughly perpendicular to both of these axes. The structural homology between N- and C-terminal domains of the @ subunit is similar to that observed in several multidomain proteins. Some examples include rhodanese (75), the aspartic proteinases (76, 77), and lactoferrin (78), all of

(H41 (S4) (H31 (S3) 130 120 110 100 90 HKTNQVLGQAL-LAK---RMGKSEII~T~~H~AS~ALLG--LKCRI~G---...

...

IIIIIIII / Ill

/ /

Ill**II*I*IIIIIIIIlIII

/

IIIIIIII

...FQRMIGEET-KAQILDKG~PDAVIACVGGGGSNAIG~~FIN-DTSVGLI~PGGH... 240

230

220

210 (H81

60 (S7)

(H9)

(S8)

(S6) (H6) (H51 (S5) 140 170 150 160 180 190 -ERQSPNVFR”- GAEVIPVHSGSATL--------------- KDACNEAZlRDWS-GS------YETAHYMLGT / III**IIII IIIIIII* / / IIIIII I*IIIIIII FP--SVG-PQHAYLNSIGRADWSITD-DEALEAFRTLC~GIIPAL-ESS~~QPE~QLL~LS310360 350320 340 330 370 (HI0 1 (S9) (HI11 (H12) (S10) FIG.7. Amino acid sequence of the j3 subunit N- and C-domains aligned according to the three-

- -

dimensional structural superposition. Residues from the N-domain (86-135 and 140-190) are shown above the structurally homologous residues from the C-domain (204-260 and 306-377). Symbols: I denotes the 65 pairs * denotes the eight amino of residues in contiguous stretches for which the Cm atoms deviate by less than 4.0 acid identities found in these stretches; / denotes pairs that also deviate by less than 4.0 A, but which are either noncontiguous or required inserted gaps in the sequence. The root-mean-square deviation of 73 Cor pairs in the contiguous regions is 2.20 Secondary structure elements are shown above and below each line; H,helices; S, strands (see Table I11 for detailed assignments).

A;

A.

Structure of the Tryptophan Synthase MultienzymeComplex which possess two roughly equal-sized domains with similar three-dimensional folds. The domains of rhodanese and the aspartic proteinases are related by pseudo-dyad axes, whereas those of lactoferrin are related by a screw-axis. The reported root-mean-square deviations in superimposed Ca atoms positions in rhodanese and the aspartyl proteases (1.95 8, for 117 Ca pairs in rhodanese (75) and 1.91 8, for 70 Ca pairs in the aspartic proteinases (76)) compare with the value of 2.2 8, reported here for 73 Ca pairs of the tryptophan synthase B subunit. Coenzyme Binding Site-Pyridoxal phosphate, the coenzyme of the p subunit of tryptophan synthase, is located at the interface between the two structural domains near the center of each p subunit (Figs. 2A, 2B, and 4C). The domains are oriented so that the pyridoxal phosphate binds near the C-terminal ends of the parallel strands of both domains. The C5’-05’-phosphate bond of pyridoxal phosphate is nearly perpendicular to theplane of the pyridine ring as in glycogen phosphorylase b (79). The phosphate group of the coenzyme is highly ligated through hydrogen bonds with the peptide backbone atoms of residues Gly-232, Gly-233, Gly-234, Ser235, and Ala-237 and with the side chains of Ser-235 and Asn236. The phosphate of the pyridoxal phosphate in glycogen phosphorylase b is also linked to 2 glycine residues. There is a highly conserved glycine rich region in several threonine dehydratases (80) which suggests that here too the phosphate of the cofactor may bind in a glycine-rich region in other pyridoxal phosphate-dependent enzymes. The negative charges on the phosphate may be neutralized by the imidazole of His-86 and the positive end of a dipole from helix 9. The pyridine ring nitrogen atom, N1, is close to the sulfhydryl of Cys-230 and within hydrogen-bonding distance with the hydroxyl of Ser-377. Although the carboxylate of Glu-350 is also near the pyridinium nitrogen atom, it is stacked parallel to the plane of the coenzyme ring with a geometry that may not favor a strong ionic interaction. Pyridoxal phosphate forms a covalent C=N (Schiff base) bond with the t-amino group of Lys-87 of the p subunit. The Schiff base configuration is “cisoid” and in a conformation that brings the Schiff base nitrogen near the 3‘-hydroxyl oxygen atom of the coenzyme. This hydroxyl may have no protein ligand although the nearby side chain of Gln-114, which is poorly localized (i.e. highly disordered), could interact with the 3’-hydroxyl or with the Schiff base nitrogen atom of pyridoxal phosphate or with both. The close proximity of the C2‘ methyl group of pyridoxal phosphate to Gly-378 may explain why Gly-378 is conserved among species; a larger residue might displace the coenzyme. Potential Active Site Residues of the @ Subunit-Although the substrate binding site of the p subunit has not yet been located by crystallographic studies using bound substrates or substrate analogs, this site must be spatially adjacent to the coenzyme binding site since Schiff base intermediates are formed between thesubstrateand coenzyme. Open space found next to the deeply buried coenzyme and lined with highly conserved residues is the likely binding site for the substrate L-serine (Fig. 4C). The t-amino group of Lys-87, which forms the Schiff base linkage in theholoenzyme and is released after L-serine binds, is ideally situated to facilitate catalysis by removing the a proton of L-serine. Although His86 seems too distant to be involved in catalysis, it may have a role in coenzyme binding. Tryptophan Synthase from Yeast and Molds-Since the sequences of tryptophan synthase from bacteria, yeast, and molds have significant homology, it can be assumed that these enzymes have similar three-dimensional structures and that an examination of the spatial arrangements of the a and p subunits in the bacterial apppcomplex would suggest how the

17867

homologous a and p regions of tryptophan synthase from yeasts and molds might be linked to form a single multifunctional polypeptide chain (81). In tryptophan synthase from Saccharomyces (82, 83) and from Neurospora (841, an Nterminal region with strong homology to the a subunit of bacteria is linked to a C-terminal region homologousto thep subunit of bacteria by a connecting stretch of about 30 amino acids. Thus, the regions in Saccharomyces and Neurospora corresponding to trpA andtrpB must be fused in theorder AB, representing a transposition of the bacterial genes which are found in theorder B-A. In theS. typhimurium tryptophan synthase, the N terminus of the p subunit is near the a l p subunit interface, while the C terminus of the a subunit is about 50 8, away on the opposite side of the a subunit (away from the a/@ interface). This may explain why a long connector with 30 amino acids is required. An even longer peptide would be required to bridge the 70 8, distance separating the C terminus of the p subunit from the N terminus of the a subunit to yield the hypothetical polypeptide resulting from a fusion of the trpB and trpA genes in the B-A orientation found in bacteria. Although a partially active fused E. coli trpB-trpA polypeptide has recently been made with a very short connector (81),our results indicate that thefusion must result in aspatialarrangement of the a and /3 subunits different from that reported here. Channeling of Indole through a Tunnel-In the a-@reaction of tryptophan synthase there is evidence that indole is not liberated as afree intermediate (7-9). A hitherto unanswered question about the mechanism of this reaction is whether the indole is transferred directly between adjacent active sites of the a and p subunits orwhether it diffuses through some kind of channel between distant active sites. Steady-state kinetics results indicate that there are separate binding sites for indole in the a and /3 subunits (7). Three mechanisms have been proposed for indole transfer (4): 1)concerted transfer between adjoining active sites; 2) covalent attachment throughout via a concerted mechanism (85); and 3) diffusion of free indole between active sites through a channel. Our finding from xray crystallogra hy that the active sites of the a and p subunits are 25 apart eliminates possibilities 1and 2 and is consistent with possibility 3. Similar conclusions have been previously reached indirectly by studies using fluorescence energy transfer (86)or cross-linking (87). An examination of the structure of the enzyme complex (Fig. 2) revealed that the two active centers in each a/@ pair are distant and areconnected by a channel or“tunnel.” This tunnel is illustrated in Fig. 8, B and C, by displaying the internal molecular surface (32) of an a/@ pair. The diameter of this tunnel is sufficient to accommodate the indole substrate. The tunnel extends from the a subunit active site through the center of interface between the p subunit N- and C-domains to the pyridoxal phosphate site in the @ subunit and beyond. Although the words “channel” and “channeling” have been used in this context, tunnel better describes its three-dimensional aspects observed here. Globular proteins are usually found to be solidly packed and interiorholes occur only rarely. The presence of such an extensive feature implies an important role for it in facilitating the a-@ reaction. It seems most likely that the tunnel functions by providing a pathway for the internal diffusion of indole between the two active sites. Most of the length of the tunnel is in the ,8 subunit since the a subunit active site is located near the surface of the a subunitand close to the a/@ subunit interface. In the a subunit, one wall of the tunnel is formed by residues 58-60, which is part of a small segment that is currently ill defined. An opposing surface of the tunnelnear the substrate binding

R

Structure of the Tryptophan S y n t h e Multienzyme Complex

17868

R

A

FIG. 8. A, smoothed backbone representation showing the folding and spatial arrangements of one a/@ subunit pair. The a subunit is blue, the @ subunit N- and C-domains are yellow and orange, respectively. Strands in thea subunit @ barrel and in sheets in both domains of the fl subunit have been highlighted in white. Indole propanol phosphate in the a subunit and pyridoxal phosphate in the fl subunit active site are shown in red. B, view of the indole tunnel between active sites showing the surface of one a/fl subunit pair (blue dot surface), the self-

Structure of the Tryptophan Synthase Multienzyme site in the a subunit is formed by side chains of residues at the C-terminal ends of the strands 2, 3, and 4 and the loops that follow. The loop containing residues 180-190 (also poorly defined at present) may form one side of the tunnel near its opening to thesolvent. The remaining two-thirds of the tunnel is buried within the (? subunit and at the interface of the Nand C-domains. The side of the tunnel formed by the Ndomain of the (? subunit is lined with residues from helix 6 and by the residues between strand 6 and helix 7, and closer to the(? active site, by residues from strand 3of the N-domain 0-sheet. Residues 17-21, near the amino terminus in the turn preceding the first helix, form the “bottom” of the tunnel in the (? subunit. The other side of the tunnel is formed from residues in the long stretch from 260-310 with random-coil conformations. Thus, the tunnel within the (3 subunit appears to be bounded on three sides by densely packed protein atoms impenetrable to indole. The “top” of the tunnel, in contrast, appears to be rather loosely covered by a single layer of long, highly mobile side chains midway along its length and would be the only point at which indole could conceivably escape. To get an appreciation for the length of the tunnel, we have packed it full of indole molecules in a head-to-tail fashion (using computer graphics) and have found that four indole molecules can be simultaneously accommodated between the substrate indole site in the a subunitand the coenzymebinding site in the (? subunit (Fig. 8C). The first of these four positions is located in the interface of the a and 0 subunits while the other three positions are found completely within the (? subunit. The fourth putative indole position is close to the pyridoxal phosphate in the (? subunit and has been tentatively assigned as theindole binding site at theactive site. Although the presence of substrates may induce global conformational changes that would affect the tunnel’s shape or effective size, the tunnel currently appears unobstructed and large enough to allow indole to diffuse throughout its length in the native, unligated form of the enzyme complex. Some points along the tunnel appear constricted and closely match the van der Waals surface of indole (Fig. 8C). Random thermal motions and “breathing” motions of the protein side chains lining the tunnel may be important for the diffusion of indole past these locations. Another interesting aspect concerns the contents of the tunnel. The outline of thetunnel shown in Fig. 8B was computed from the refined atomic positions of the protein atoms. Although all the known protein atoms in these areas have been accounted for in the electron density map, several weak density features remain in the tunnel andsuggest that the tunnelis occupied by looselybound water molecules or by solution components such as spermine. It is possible that the precise shape and dimensions of the tunnel may change as the conformations and positions of the protein atoms lining the tunnel are improved during further refinement at increased resolution. A visual examination of the arrangement of the tunnel and the two active sites suggests the following overall action in catalysis: indole glycerol phosphate substrate enters through an opening into the a subunit active site, the “front door,”

Complex

17869

and theglyceraldehyde 3-phosphate leaves via the same route. Similarly, in the /3 subunit, L-serine enters and L-tryptophan exits via another opening to the solvent, another front door. The a and (? active sites also each have a “back door” with the tunnel acting to connect the two back doors. The tunnel functions by allowing indole to diffuse between the back doors of the two active sites and prevents itsescape to thesolvent. The channeling of metabolic intermediates between the active sites of enzymes has many advantages (4, 88). Channeling can prevent loss of intermediates by diffusion, protect chemically labile intermediates, and decrease transit time. The probability (orrate) of a reaction can be increased immensely by converting the diffusion and binding of intermediates to anessentially one-dimensional process. Channeling may be particularly important in the case of indole since this molecule is nonpolar and may escape the cell through its membranes (90). Recent kinetic studies suggest that free indole is also channeled through the a subunit to the active site of the (? subunit in the (? reaction (89). CONCLUSIONS AND FUTURE DIRECTIONS

There is now compelling evidence that much of intermediary metabolism occurs in organized states which may entail protein-protein complexes (88).While many of these protein interactions are transient or weak (and therefore difficult to demonstrate experimentally), some can be quite tight as in the tryptophan synthase complex. Interactions between sequential enzymes in the cell are believed to provide unique kinetic and catalytic properties which differ from the counterpart systems in dilute solution (88).Channeling of the intermediates of these reactions has been reported for many systems and reviewed extensively (4, 88, 91). The observed association of the a and (? subunits of tryptophan synthase and the demonstration of a tunnel between two active sites representsan unexpected and at present unique mechanism for such channeling. It will be interesting to see whether similar mechanisms haveevolved in other enzyme systems. One important property of tryptophan synthase which has not been clarified by the present studies is the mechanism of the mutual activation of the a and (? subunits upon complex formation. Evidence that this activation is the result of a conformational change in one or bothsubunitshas been previously obtained from physical and kinetic studies (29, 30, 92-94). A comparison of the three-dimensional structures of the individual a and (? subunits with that of the a& complex would be required in order to explore this possibility. The determination of the crystal structure is usually only the first step in the structuralanalysis of a protein. Currentiy this enzyme system is being refined to a resolution of 2.2 A, at which point we plan to investigate further its mechanism of actionthrough the analysis of inhibitor binding and through site-directed mutagenesis. The crystals of the enzyme described here have been shown to be catalytically active in the a , (?, and a-(?reactions (13), so that both active sites are

-

complimentary surface formingthe /3 subunit dimer interface (gold),and the buried contact surface between a and /3 subunits (orange). The portion of the molecular surface buried deeply within the protein (highlighted in white) outlines the edges of the indole tunnel. The tunnel leads from the a subunit active site (at the lower left, with indole propanol phosphate in a red dot surface) to the pyridoxal phosphate at the active site of the /3 subunit (near the upper right, also in a red dot surface). C, enlarged view of the indole tunnel, as shown in B. The tunnel is shown packed with indole molecules (yellow dot surfaces) between indole propanol phosphate (lower left, red surface) and pyridoxal phosphate (upper right, also in a red surface). The buried a/@ subunit contact surface is shown in green. An empty pocket seen to the right of indole propanol phosphate has been tentatively identified as the “effector” indole site of the a subunit. The four indole molecules were positioned on the basis of computer graphics modeling and are intended only to illustrate the size and shape of the tunnel.

17870

Structure of the Tryptophan Synthase Multienzyme

Complex

accessible to substrate. The rich background ofinvestigations 30. Wilhelm, P., Pilz, I., Lane, A. N., and Kirschner, K. (1982) Eur. J. Biochem. 129,51-56 on the genetics of this system in E. coli provides us with a K., May, R. P., Kirschner, K., Lane, A. N., Szadkowski, H., number of clear suggestions as to which residues will be the 31. Ibel, Dauvergne,M.T.,andZulauf, M. (1985) Eur. J. Biochem. 1 6 1 , most likely to yieldrelevantinformation by site-directed 505-514 mutagenesis. In addition some of the second site revertants 32. Connolly, M. L. (1983) J. Appl. Crystallogr. 16,548-558 will be examined in order to understand their effect. Finally 33. Case, D. A., and Karplus, M. (1979) J. Mol. Biol. 132,343-368 we intend to examine the effect of mutations that would have 34. Silva, J. L., Miles, E. W., and Weber, G. (1986) Biochemistry 2 5 , 5780-5786 the potential to block the tunnel in order to observe their 35. Sheriff, S., Silverton, E. W., Padlan, E. A., Cohen, G. H., Smitheffect on the catalysis. Gill, S. J., Finzel, B. C., and Davies, D. R. (1987) Proc. Natl. Acknowledgments-We thank Ronald Bauerle for the gift of the Salmonella strain, Kasper Kirschner for the gift of indole propanol phosphate, Joseph Shiloach for the growth of bacteria, Ron Chandross, Randy Rains, Robert Kretsinger, and Ng. H. Xuong and colleagues at University of California at San Diego for assistance with area detector data collection, B. C. Wang and G. H. Cohen for use of their software, and JoanHanley-Hyde for carefully reading the manuscript.

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