Affinity Labeling, Molecular Cloning, and ... - Wiley Online Library

PART I. STRUCTURE AND MOLECULAR BIOLOGY (SEX STEROID-BINDING PROTEIN, COR TICOSTEROID-BINDING GLOBULIW AND ANDROGENBINDING PROTEIN)

Affinity Labeling, Molecular Cloning, and Comparative Amino Acid Sequence Analyses of Sex Steroid-Binding Protein of Plasma A Multidisciplinary Approach for Understanding Steroid-Protein Interaction and Its Physiological Role" PHILIP H. PETRA~"BENITO G. QUE,~"PEARL c. NAMKUNG,~J. B. A. ROSS,~HARRY CHARBONNEAU,' KENNETH A. WALSH,' PATRICK R. GRIFFIN,' JEFFREY SHABANOWITZ,' AND DONALD F. HUNT Departments of

Obstetrics and Gynecology and 'Biochemistry University of Washington Seattle, Washington 98195 Department of Biochemistry Mount Sinai School of Medicine New York, New York 10029 and Department of Chemistry University of Virginia Charlottesville, Virginia 22901

Steroid-protein interaction is recognized as an important biochemical process integral in the expression of steroid hormonal activity. This is indicated by the existence of special extracellular and intracellular noncatalytic steroid-binding proteins that par'This research was supported by National Institutes of Health Grants HD-13956 (PHP), GM-37537 (DFH), GM-15731 (KAW),and HD-17542 (JBAR). Instrument development funds were awarded to DFH by the Monsanto Co. and the National Science Foundation (Grant CHE8319728). 10

PETRA et 01.: AFFINITY LABELING OF SBP

11

ticipate in the transport of steroid hormones from plasma to the nuclei of target cells. The nature of steroid-protein interaction, however, is not well understood to this day because it was not until recently that purification and characterization of a few steroidbinding proteins, particularly those in plasma, was possible. This has opened the door to a fruitful period in the expansion of knowledge in this field that should culminate in an understanding of processes whereby steroid hormones are recognized and selected for eventual expression in target cells. Understanding the interaction between steroids and proteins is likely to be difficult. Although the chemical structure of steroid hormones has been known for many years, that of proteins is still evolving. Every protein is different and although the field has progressed significantly in recent years, we still do not understand the processes which allow formation of the three-dimensional structure of a protein from the linear arrangement of its amino acid residues in the polypeptide chain. Since the nature of forces holding a protein together are similar to those which control the interaction of proteins with their ligands, progress in understanding steroid-protein interaction should be directly related to the folding of proteins. Solution of the problem will require a multidisciplinary approach. Biochemical studies must be accompanied by X-ray diffraction analysis, molecular cloning, and physiological studies. To this end, this laboratory has undertaken characterization of SBP, the sex steroid-binding protein of plasma (also termed SHBG), with the ultimate goal of identifying the steroid-binding site. In this paper we briefly describe a variety of approaches under present use and present some preliminary data to illustrate and emphasize the importance of a multidisciplinary strategy towards the understanding of steroid-protein interaction.

AN HYPOTHETICAL REPRESENTATION OF THE STEROIDBINDING SITE OF HUMAN SBP BASED ON ITS AMINO ACID SEQUENCE Native human SBP is a dimer' composed of two identical subunits of molecular weights of about 46,700.*~'The dimer binds one molecule of steroid.' The amino acid sequence of human SBP, determined two years ago by protein sequencing methods: 1. The SBP monomer is composed of a single polypeptide chain is shown in FIGURE having 373 amino acids, 3 oligosaccharide chains, and 2 disulfide bonds. There are two N-linked sugar chains located at Asn-351 and Asn-367, and one 0-linked sugar chain at Thr-7. Only one tyrosine residue is present, located at position 57. A predominantly hydrophobic region containing alternating leucine residues exists beginning at Trp-247 and ending at Gly-291. Located within that region is an internal amino acid sequence repeat, shown more clearly in FIGURE2, starting from Leu-248 to Gly262 and repeated from Leu-277 to Gly-291. The polypeptide segment joining the repeated sequence contains three proline residues at positions 265, 270, and 276. These data suggest that the hydrophobic region containing the internal sequence repeat might represent the general feature of the steroid-binding site. Since the binding data indicate interaction with one molecule of steroid,' we have the inherent problem of describing a model having a molecular arrangement satisfying the binding stoichiometry of one asymmetric ligand, such as the steroid, to a homodimeric protein. One possibility is that formation of the steroid-protein complex occurs when the first half of the sequence repeat of one monomer interacts with one face of the steroid

rl

10

Leu-Arg-Pro-Val-Leu-ProThr Gln-Ser-Ala-His-Asp-Pro-Pro-Ala 20

30

Val -His-Leu-Ser- A m - G l y - P r o - G l y - G l n - G l u - Pro-Ile -Ala-Val-Met 40

Thr-Phe-Asp-Leu-Thr-Lys-Ile-Thr-Lys-Thr-Ser-Ser-Ser-Phe-Glu 50

60

Val-Arg-Thr-Trp-Asp-Pro-Glu-Gly-Val-Ile-Phe 70

Asn-Pro-Lys-Asp-Asp-Trp-Phe-Met-Leu-Gly-Leu-Arg-Asp-Gly-Arg 80

90

Pro-Glu-Ile-Gln-Leu-His-Asn-His-Trp-Ala-Gln-Leu-Thr-Val-Gly 100 Ala-Gly-Pro-Arg-Leu-Asp-Asp-Gly-Arg-Trp-His-Gln-Val-Glu-Val 120

110

Lys-Met-Glu-Gly-Asp-Ser-Val-Leu-Leu-Glu-Val-Asp-Gly-Glu-Glu 130 Val-Leu-Arg-Leu-Arg-Gln-Val-Ser-Gly-Pro-Leu-Thr-Ser-Lys-Arg 140

150

His-Pro-Ile-Met-Arg-Ile-Ala-Leu-Gly-Gly-Leu-Leu-Phe-Pro-Ala

170

180

200

210

Ile-Phe-Leu-Pro-Pro-Gly-Thr-Gln-Ala-Glu-Phe-Asn-Leu-Arg-Asp 220

Ile-Pro-Gln-Pro-His-Ala-Glu-Pro-Trp-Ala-Phe-Ser-Leu-Asp-Leu 2 30 240 Gly-Leu-Lys-Gln-Ala-Ala-Gly-Ser-Gly-His-Leu-Leu-Ala-Leu-Gly 250

Thr-Pro-Glu-Asn-Pro-SerT r p - L e u - S e r - L e u - H i s - L e u - G l n - A s p - G l n 2 70’

260

[Lys-Val-Val-Leu-Ser-Ser-Gly-Ser-Gly-Pro-Gly-Le~-Asp-Leu-Pro~ oon LOU

[Leu-Val-Leu-Gly-Leu-Pro-Leu-Gln-Leu-Lys-leu-Ser-Met-Ser-Ar~J 3911 snn - _IVal-Val-Leu-Ser-Gln-GlyjSer-Lys-Met-Lys-Ala-Leu-Ala-Leu-Pro 310 Pro-Leu-Gly-Leu-Ala-Pro-Leu-Leu-Asn-Leu-Trp-ALa-Lys-Pro-Gln

---

320

330

Gly-Arg-Leu-Phe-Leu-Gly-Ala-Leu-Pro-Gly-Glu-Asp-Ser-Ser-Thr 340 360

370

FIGURE 1. Amino acid sequence of human SBP.’ The hydrophobic core is located at residues 241 through 291.

12

13

PETRA el of.: AFFINITY LABELING OF SBP

while the second half of the repeat of the second monomer interacts with the other face. As a result, a conformation change in the protein could occur preventing the other set of sequence repeat from folding together to form a second steroid-binding site. In this way, binding of the asymmetric steroid might be explained since the repeated sequences are not exact duplicates. We have recently obtained data to support such a model which is characteristic of a negative cooperative mechanism of binding.’ Measurement of stoichiometry of steroid binding after storage of the pure protein in the presence of steroid at 4 “C (or in the frozen state) for months, shows two moles of steroid bound per mole of dimer. This would suggest that storage of the pure protein abolishes negative cooperatively through a conformation change that allows the second set of sequence repeat to form the other steroid-binding site. According to that model the internal sequence repeat would form the essential feature of the steroid-binding site. Amino acid side-chains coming from other regions of the molecule could then be brought together and interact with the steroid to provide specificity resulting in the formation of a “steroid sandwich” as recently postulated.”’

L T N A V S A I T

L P S C

R F P G L N P P P V L F P

P D K P R L T Q E L G C Q

V L D R L R S P N G L L S

L T D L R L L H P L A N P

P K W D Q P R A

T I F D V L S E f

Q T M G S V C P

S K L R G P D W l

A T G W P A V A

P P L L N G L W A G N G T

K L Q D

H S L H L L E F T L W G A

D P P A V H L S N G P G Q E P I A V M S S F E V R T W D P E G V I F Y G D T R D G R P E I Q L H N H W A Q L T V G Q V E V K M E G D S V L L E V D G E E T S K R H P I M R I A L G G L L F P A D G C L R R D S W L D K Q A E I S A S S N P G I F L P P G T Q A E F N L R D S L D L G L K A A G S G H L L A L G DX-a G S G a G L D L a M S V V L K M K A L A L P A K P Q G R L F L G A L P G E D S S T Q R L D V D Q A L N R S H E I W T H S S H

30 60

90 120 150 180 210 240

270 300 330 360

FIGURE 2. Amino acid sequence of human SBP indicating the location of the internal amino acid repeat.

Although assignment of specific regions of the hydrophobic core to formation of the steroid-binding site is slightly different from that proposed in our earlier publications’~’ (detection of the sequence repeat was made while those were in press ), the “sandwich” idea is essentially the same. Although the negative cooperative model proposed above indicates that the subunits would each contribute one half of the sequence repeat to form the full potential binding site, one subunit could also in theory contribute both repeats to form the site. Presence of proline residues 265, 270, and 276 in the segment connecting the sequence repeat would produce the “turn” resulting in a “wrapped around” configuration of the polypeptide chain bringing the two halves of the repeat together to form the site. In that case, the presumed conformation change resulting from steroid binding would prevent the other subunit from binding steroid. Although that version of the model describes the steroid-binding site as being formed through a subunit intramolecular arrangement, the symmetry and stoichiometry of binding are still satisfied. Therefore, whether the structural arrangement is either intramolecular or intermolecular, both models essentially describe the same steroid-binding site. It should be emphasized, however, that the hydrophobic region could also serve as a

14

ANNALS NEW YORK ACADEMY OF SCIENCES

general stabilizing feature for the native conformation of the molecule and therefore may have nothing to do with steroid binding. That interpretation cannot be ruled out at this time and the purpose of the experiments under consideration is to provide the necessary data to support the proposal.

THE AFFINITY LABELING APPROACH To test the hypothesis presented above we are attempting to identify amino acid residues within the steroid-binding site and place them in the amino acid sequence. The experimental method of choice for that approach is affinity labeling. SBP is reacted with radioactive derivatives of DHT, T, or E, containing chromophores or chemically reactive groups. Since the derivatives are structurally related to the native ligands, they should bind within the steroid-binding site. The chemical reactivity of the groups is such that the derivatives should attach covalently to amino acid sidechains within the binding site thereby labeling them for identification. The next steps are to digest the labeled SBP with proteases, isolate and sequence the radioactive peptides containing the label, and place the identified amino acid residue within the amino acid sequence. By using several derivatives with reactive groups at different positions within the steroid, one should build a catalog of labeled amino acid residues 3 shows examples which can be used to "reconstruct" the steroid binding site. FIGURE of affinity labels we are using to characterize the steroid-binding sites of human and rabbit SBPs. Equilenin, an estrogen isolated from horse urine, was found to compete with testosterone for the binding site of partially-purified SBP.6 It is of special interest because of the aromatic structure of rings A and B is identical to 2-naphthol, a strongly fluorescent molecule with well-characterized excited state properties.' Its binding affinity for pure human and rabbit SBPs is high with Kds of 1.7 x lo-* M and 5.2 x 10 -* M, respe~tively.'.~Results of fluorescence excitation and quenching experiments with collisional quenchers KI and acrylamide, indicate that the bound steroid has limited accessibility to the bulk solvent and that there are no anionic surface groups near the steroid-binding site? Fluorescence excitation spectra of the SBPequilenin complex are similar to the absorption spectra of equilenin in low-dielectric solvents. Fluorescence emission of the equilenin complex, however, exhibits wavelength shifts (red shifts) opposite to those of the steroid in low-dielectric solvents (blue shifts) but similar to the red shifts produced by the addition of the proton acceptor triethylamine to equilenin in cyclohexane? Those data suggest that the steroid binding sites of human SBP and rabbit SBP are nonpolar cavities containing a proton acceptor that participates in steroid binding possibly through hydrogen bond formation with the %OX0 group of the steroid. This finding will be useful for final interpretation of binding data resulting from other experiments. Bromoacetate steroid derivatives, including DHT-17P-bromoacetate shown in FIGURE 3, were used previously for labeling steroid-binding sites. Ganguly and Warren'' and later Sweet and Samant" used them for specific modification of the active site of 3a,2OP-hydroxysteroid dehydrogenase. We recently found that DHT- 17P-bromoacetate specifically inactivates SBP by interacting with the steroid-binding site (Namkung and Petra, to be published). Isolation of peptides is in progress. Inactivation of SBP

1s

PETRA ef al.: AFFINITY LABELING OF SBP

with the dibromo derivatives of DHT, shown in FIGURE 3, will hopefully cross-link the steroid-binding site by interacting with two different amino acid residues and thus provide information on the distance separating them. A6-Testosteroneis a photoaffinity label that has been used for labeling rat ABP" and rabbit" and human SBPS.'~The purpose of our experiments is to identify the amino acid residue(s) in the steroid-binding site which are modified when SBP is photolabeled in its presence. The experiment consists of treating SBP (0.2 nmoles in

Equilenin

DHT-178-bromoacetate

A6-Testosterone

DHT-38, 178-bromoacetate

FIGURE 3. Affinity labels of human SBP. Equilenin, d-3-hydroxy-1,3,5(10),6,8-estrapentaenDHT- 1P-bromoacetate, Sa-androstan17-one; A6-Testosterone, 4,6-androstadien-l7fi-ol-3-one; 17~-bromoacetyloxy-3-one; DHT-3P-l7/3-bromoacetate, Sa-androstan-3~,17fi-dibromoacetyloxy.

0.5 rnl of 10 mM Tris-C1, pH 7.4) with [ 'H]A6-T at 0 "C for 30 min at wavelength > 300 nm in the presence or absence of 100-fold molar excess of radioinert DHT. As shown in FIGURE 4, photoaffinity labeling of the pure protein results in the formation of radioactive bands on SDS-PAGE corresponding to those characteristic of pure SBP. Presence of DHT during photolysis prevents A6-T labeling as shown by the absence of the radioactive bands. The data therefore indicate that specific and irreversible binding of the label has occurred within the steroid-binding site. Modified SBP was then digested with Achrornobacter protease I (which catalyses cleavage only at lysine residues). The products were reduced and alkylated with iodoacetic acid,


16

10

20 Gel Slices

30

40

FIGURE 4. Electrophoresis of [ 'H]A6-testosterone-labeledSBP polyacrylamide gels according to Petra et al.*' ( 0 -- - - 0 )Photolysis in the absence of DHT. ( 0 -- - - 0 )Photolysis in the presence of 200-fold molar excess of DHT.The inset shows two gels, one containing molecular weight standards and the other labeled human SBP.

and then fractionated by filtration on tandem TSK-2000SW columns equilibrated in 6M Gdn-HC1. Two radioactive fractions were examined in more detail. The one containing the greatest percentage of radioactivity was further purified by reverse phase HPLC and sequenced. The data suggest that the labeled amino acid is associated with a large carboxy-terminal peptide beginning at Ala-296. Although the peptide was sequenced for about 25 turns by Edman degradation and was shown to be completely identical to the published sequence, the labeled amino acid residue was not recovered as the product of a single cycle. Assignment must therefore await further sequence analysis (work in progress). Similar results were reported recently by Hammond and co-~orkers.'~ Those authors also were not able to identify the amino acid residue(s) containing the A6-T label and concluded similarly that the labeled site was located at the carboxy-terminal end of SBP. It is therefore likely that the carboxyterminal region of human SBP contains an amino acid residue which is present in the steroid-binding site. However, this does not mean that the carboxy-terminal end of the protein necessarily represents the steroid-binding domain. The steroid-binding site may well be brought together from a number of amino acid side-chains located in various sequences of the molecule. Chemical modification using other affinity labels will help in further defining the structural features of the steroid-binding site.

PETRA el 01.: AFFINITY LABELING OF SBP

17

MOLECULAR CLONING Another approach to gain insights into the structural basis of steroid-protein interaction is through molecular cloning and cDNA expression in appropriate vectors 5. either in bacteria or mammalian cell systems, as shown schematically in FIGURE For that purpose it is advantageous to have cDNA clones of varying insert sizes, including one of full length. In this way, it becomes possible to correlate the minimum size of coding sequence required for generating DHT-binding activity. Such data allow analysis of the question whether the entire sequence of SBP is necessary for steroidbinding. Furthermore, through site-directed mutagenesis of the cDNA, we expect to identify specific amino acids that participate in steroid binding. Data obtained from affinity labeling are valuable in designing such experiments. Although the approach is very different from that described above for affinity labeling, the two are complementary and produce similar end results. The advantage of in vitro site-directed mutagenesis is that closely related amino acid residues can be replaced at specific positions in the polypeptide chain with little perturbation to be expected in protein conformation. In contrast, introduction of affinity labels or other groups into the steroid-binding site may result in changes of local conformation, leading to erroneous identification of residues at the native site. This classic caveat of all protein modification studies is avoided by using site-directed mutagenesis for the study of protein structure and such an approach is now enthusiastically taken by protein chemists. Molecular cloning involves isolation of positive hgtl1 recombinant phage containing cDNA coding sequences for SBP. Since the SBP message level is relatively low in adult human liver and HepG2 cells (unpublished observations), isolation of cDNAs requires screening of millions of clones. Fortunately, Joseph and co-workers,’6 while working on rat ABP cloning, isolated a positive hgt 11 phage clone from a fetal human liver cDNA library using rat ABP cDNA as probe. The cDNA insert was isolated, sequenced, and found to contain two thirds of the coding sequence of mature 6. The cDNA contains a coding region of 843 human SBP” as shown in FIGURE nucleotides for SBP starting at Gly-92 and continuing through the carboxyl-end residue His-273, followed by a stop codon (TAA), a short 3’ untranslated region of 19 nucleotides, and a polydA tail of 49 nucleotides. A potential polyadenylation signal sequence (ATTAAA) is located as part of the coding region of the carboxy-terminal histidine residue and the termination codon. Similar polyadenylation sites have been found as part of termination codons in the case of the &subunit of hCG1*and blood

SBp

wild type

wild type

I sile-di;ecled

FIGURE 5. Experimental strategy for characterizing the steroid-binding site of SBP by recombinant DNA techniques.

muragtnesis

J

c DNA mutant

expression

SBP mutant

T P E N P S W L S L H L Q D Q ACA CCA GAG AAC CCA TCT TGG CTC AGT CTC CAC CTC CAA GAT CAA

PETRA er al.: AFFINITY LABELING OF SBP

19

clotting Factor X.I9The cDNA contains the coding region for the hydrophobic region (including the amino acid sequence repeat) described above (FIGURE2). It will be interesting to express that cDNA clone and test for steroid-binding activity. We recently isolated (using immunodetection as de~cribed’~) and sequenced three SBP cDNAs from a hgtl 1 cDNA expression library prepared from HepG2 cells (Que et al., to be published). The polyadenylation signal sequence ATTAAA of one of the clones was also found to be located within the termination codon as in the case of the liver cDNA. That canonical sequence might be functional and could serve in the regulation of SBP gene expression. All the deduced Two other laboratories have published SBP cDNA sequences are in agreement with the published amino acid sequence of SBP with one interesting difference. Gershagen and co-workers” isolated a partial cDNA clone and found that the following deduced sequence at the amino terminal region: VHSAAQTTLIAVMTF.. . . The first nine residues of this sequence are different from those beginning at residue 18 in the protein (FIGURE1) although the remaining sequence through the carboxyl terminus and the 3’ untranslated region is in complete agreement with our results (see FIGURES 1 and 6). Since the nucleotide sequence shown in FIGURE 6 starts at Gly-92, we were not able to confirm their finding; however, Hammond and c o - w o r k e r ~ ’did ~ not find that sequence in their cDNA clone. An explanation of this discrepancy must await determination of the nucleotide sequence of the SBP gene.

COMPARATIVE AMINO ACID SEQUENCE ANALYSES Structural comparison of homologous and functionally-related proteins has often lead to insights into their mechanisms of action and biological roles. Two years ago, the first structural relationship between two extracellular steroid-binding proteins was discovered jointly by Dr. Joseph’s laboratory and our own?’ We established that rat ABP and human SBP were homologous proteins by showing that 68% of their amino acid residues were identical in their sequential arrangement without the inclusion of deletions or insertions. That finding coupled with their biochemical similarities strongly suggest that in organisms that contain both proteins, such as in humans and in rabbits, the proteins are likely to be identical possibly with some variations in their sugar Although the relationship between ABP and SBP and their genes is not yet clear, such a finding would strongly suggest that the same protein is synthesized in different tissues but under different regulatory mechanisms. An understanding of the molecular basis of their regulation would follow by studying the 5‘-flanking control region of the ABP and SBP genes where structural differences would be expected. Since we and others had purified and partially characterized SBPs from other species, namely, rabbit SBP,22.2’.24bovine SBP,” canine SBP,26 monkey SBP,4 and baboon SBP,22the homology between rat ABP and human SBP led us to re-examine some of those SBPs and compare them to the human protein. Our most recent work has concentrated on the comparison of human and rabbit SBPs. Although the two proteins are very similar to each 0ther,2’-’~they differ significantly in their steroidbinding specificities, where rabbit SBP is primarily an androgen-binding protein with little affinity for 17/3-e~tradiol.~~ That finding makes comparison of those two proteins likely to yield new information about the molecular basis of steroid-binding specificity. 7, the only structural difference between E, and T resides in As shown in FIGURE

20


ring A. Therefore, comparison of the steroid-binding sites of human and rabbit SBPs where ring A is recognized should reveal the protein structural elements that distinguish between estrogen and androgen binding.' Furthermore, scanning the sequences in looking for areas of similarities and differences is also likely to identify possible functional regions and therefore help in the design of affinity labeling and site-directed mutagenesis experiments. To this end, in collaboration with Dr. Donald Hunt and his co-workers at the University of Virginia, we have determined the amino acid sequence of rabbit SBP." 8. As expected, The data are compared with human SBP and rat ABP in FIGURE the data indicate that rabbit SBP is homologous to the other steroid binding proteins. Rabbit SBP is more similar to human SBP because approximately 75% of its amino acid residues are identical to those of human SBP but only 68% to those of rat ABP. Turning to the hydrophobic region, the first half of the sequence repeat of both human and rabbit SBPs (residues 248 to 262) are identical, whereas 4 residues are different in the second half (residues 282, 283, 285, 286). In the case of rat ABP, there are two changes in the first half of the repeat (residues 249 and 256) and five changes in

Testosterone FIGURE 7. Schematic representation of the three-dimensional structures of testosterone and 17/3-estradiol.

170-Estradiol

the second half (residues 282, 283, 284, 285, 287) when compared to human SBP. In fact, the sequence between residues 282 through 287 in the second half of the repeat is the most variable in the hydrophobic core of all three proteins as shown in FIGURE 8. If, as postulated, that region is involved in steroid binding, its structural variability may be a reflection of the specificity differences characteristic of human and rabbit SBPs discussed above. That polypeptide segment is certainly a logical place for designing oligo-directed mutagenesis experiments. When the amino acid sequence of human SBP was determined in late 1985, no homology was found to any other protein sequence existing at the time in data banks5 Shortly after discovery of homology between rat ABP and human SBP reported at the First International Symposium on Steroid Binding Proteins,2' Baker and coworkers*' made the interesting observation that rat ABP was homologous to the carboxy-terminal domain of bovine protein S which had just been sequenced?' Protein S, a vitamin K-dependent is thought to function in blood clotting by serving as co-factor for activated protein C, a specific protease which degrades Factors Va and VIIIa.30.3'It also appears to have some role in the regulation of the complement

21

PETRA et al.: AFFINITY LABELING OF SBP

*

20

10

249 50

F E V R T W D P E G V I F Y G D T N P K D D W F M L G 60

80

70

F E L R T W D ~ E G V I F Y G D T N P K D D W F M L G F E F R T W D P E G V I F Y G D T N JJF R T ~ D G v~ I E ~ E s IA 100

90

pl

L05uat

P T S P

L L L L

T H A F

S K R H P I M R I A L G Z D K P Q P V M @ I A m G G L L S M R I A L G G D H P K P E N G L L E T K V Y F

L L L A

L F L G

F P L F

P - - - P - - P K R V

A P T S

190

S N L R L P L V P A L D G C L R R D S S S L R L P L V P A L D G C L R R G S S K L R F P L V P A L D G C I R R D I E E L I K P I N P R L D G C I R S W N 200

210

220

310

*

320

370

IP G N G T D A S HI

HUMAN SBP

FIGURE 8. Degree of sequence similarity between human SBP,’ rabbit SBP,” rat ABP,” and human protein S.3J.uThe alignment of human protein S was determined as described in TABLE 1. The dashes represent sequence-gaps introduced by the computer analysis.


22

system by interacting noncovalently with regulatory complement protein C4b-binding protein.’* However, unlike all the other vitamin K-dependent clotting factors, the carboxy-terminal domain of protein S is not a serine protease. Recent determination of the amino acid sequence of human protein S”.34 prompted us to compare its carboxy-terminal domain to human SBP.” Using the ALIGN 8 and TABLE1 reveal a program of Dayhoff et &I5 the results shown in FIGURE similarity in sequence with the likelihood that the two proteins diverged from a common ancestor. 8 was The illustrated alignment between Protein S and human SBP in FIGURE determined by comparison with those derived from random sequences of the same composition. The alignment score is 17.3 standard deviation units from the mean of 100 random alignment scores indicating a high degree of confidence in the significance

TABLE I. Degree of Similarity between Human SBP, Human Protein S (PS), and

Rat ABP Human SBP Human SBP Human PS

17.31d

Rat ABP

54.72d

Human PS

Rat ABP

(88/364)”[ 8]* 24.2%‘

(255/373)”[0jb 68.4%‘ (92/365 )”[9Ib 25.2%‘

16.88d

Identities/possible matches: the number of identical residues between two sequences is compared with the total possible matches between residues using the ALIGN program with the Mutation Data Matrix]’ and a penalty for a gap (break) = 16. [ ] number of breaks. Percent identity. Evolutionary distance in PAM’S (computed from percent differences). PAM = accepted point mutation (rearranged acronym) considered a measure of the amount of evolutionary change.]’

of the structural relationship. The degree of similarity is spread throughout the two molecules with the inclusion of 8 gaps. Although the structural relationship between the two proteins suggests a possible role of sex steroids in blood clotting, at this time there are no data to support that idea and the biological significance of the homology must wait for further experimentation. TABLE1 also shows a high degree of similarity between the carboxy-terminal domain of human protein S and rat ABP (16.9 units) and, as shown previously, between human SBP and rat ABP (54.7 units). In summary, this paper describes several experimental approaches towards understanding the steroid-binding site of SBP. These efforts, in concert with those from other laboratories, should bring us closer to defining the molecular basis of steroidprotein interaction. The biochemical information is now available for designing experiments to probe the role of plasma steroid-binding proteins in the action of steroid hormones.

PETRA er al.: AFFINITY LABELING OF S B P

23

ACKNOWLEDGMENTS The authors would like to thank Drs. Gaidano, Frairia, Berta, and Fortunati for their invitation to present this paper, Dr. Fred Hagen of Zymogenetics (Seattle) for providing the HepG2 cDNA library, Ms. Santosh Kumar for her contribution in peptide purification work, and Mr. Lowell Ericsson for computer analyses of the comparison between amino acid sequences.

REFERENCES

I. 2. 3. 4. 5.

6.

7. 8. 9.

10. 11.

12. 13. 14. 15. 16.

17.

18. 19. 20. 21. 22. 23. 24. 25. 26.

PETRA,P. H. 1979. J. Ster. Biochem. 11: 245-262. PETRA,P. H., S . KUMAR,R. HAYES,L. H. ERICSSON & K. TITANI.1986. J. Ster. Biochem. 24: 45-49. PETRA,P. H., P. C. NAMKUNG, K. TITANI& K. A. WALSH.1986. In Binding Proteins of Steroid Hormones. M. G. Forest & M. Pugeat, Eds. Colloque/INSERM. Vol. 149: 15-30. John Libbey Eurotext. London /Paris. TURNER,E., J. B. A. ROSS, P. C. NAMKUNG & P. H. PETRA. 1984. Biochemistry 23: 492-497. WALSH,K. A,, K. TITANI,S . KUMAR,R. HAYES& P. H. PETRA. 1986. Biochemistry 2 5 7584-7590. LATA,G. F., H. K. Hu, G. BAGSHAW& R. F. TUCKER.1980. Arch. Biochem. Biophys. 199: 220-227. LAWS,W. R. & L. BRAND.1979. J. Phys. Chem. 8 3 795-799. Ross, J. B. A., R. TORRES& P. H. PETRA. 1982. FEBS Lett. 1 4 9 240-244. ORSTAN,A., M. F. LULKA,B. EIDE, P. H. PETRA& J. B. A. ROSS. 1986. Biochemistry 2 5 2586-2692. GANGULY, M. & J. C. WARREN.1971. J. Biol. Chem. 246: 3646-3652. SWEET,F. & B. R. SAMANT.1980. Biochemistry 19: 978-986. TAYLOR,C. A., H. E. SMITH& B. J. DANZO.1980. Proc. Natl. Acad. Sci. USA 77: 234-238. DANZO,B. J., C. A. TAYLOR& B. C. ELLER.1982. Endocrinology 111: 1278-1285. CHENG,S. L., N. KOTITE& N. A. MUSTO. 1984. J. Ster. Biochem. 21: 669-676. HAMMOND, G. L., D. A. UNDERHILL, C. L. SMITH,I. S. GOPING, M. J. HARLEY,N. A. MUSTO,C. Y. CHENG& C. W. BARDIN.1987. FEBS Lett. 215: 100-104. JOSEPH,D. R., S . H. HALL& F. S . FRENCH. 1986. In Binding Proteins of Steroid Hormones. M. G. Forest & M. Pugeat, Eds. Colloque/INSERM. Vol. 149: 123-135. John Libbey Eurotext . London / Paris. QUE, B. G. & P. H. PETRA. 1987. FEBS Lett. 2 1 9 405-409. FIDDES,J. C. & H. M. GOODMAN.1980. Nature 286 684-687. LEYTUS,S . P., D. W. CHUNG,W. KIESEL,K. KURACHI & E. W. DAVIE.1984. Proc. Natl. Sci. USA 81: 3699-3702. GERSHAGEN, S., P. FERNLUND & A. LUNDWALL. 1987. FEBS Lett. 2 2 0 129- 135. PETRA,P. H., K. TITANI,K. A. WALSH,D. R. JOSEPH,S . H. HALL& F. S. FRENCH. 1986. In Proteins of Steroid Hormones. M. G. Forest & M. Pugeat, Eds. Colloque/ INSERM. Vol. 149: 137- 142. John Libbey Eurotext. London/Paris. PETRA,P. H., P. C. NAMKUNG, D. F. SENEAR,D. A. MCCRAE,K. W. ROUSSLANG, D. C. TELLER& J. B. A. Ross. 1986. J. Ster. Biochem. 25: 191-200. MICKELSON, K. E. & P. H.PETRA. 1978. J. Biol. Chem. 2 5 3 5293-5298. KOTITE,N. J. & N. A. MUSTO. 1982. J. Biol. Chem. 257: 5118-5123. SUZUKI,Y., E. ITAGAKI,H. MORI& T. HOSAYA.1977. J. Biochem. 81: 1721-1731. SUZUKI,Y., Y. OKUMURA & H. SINOHARA. 1979. J. Biochem. 85: 1195-1203.

24


27. 28.

BAKER,M. E., F. S. FRENCH& D. R. JOSEPH.1987. Biochem. J. 243: 293-296. DAHLBACK, B., A. LUNDWALL & J. STENFLO.1986. Proc. Natl. Acad. Sci. USA 83:

29. 30. 31.

DISCIPIO,R. G. & E. W. DAVIE.1979. Biochemistry 18: 899-904. WALKER,F. J. 1981. J. Biol. Chem. 2 5 6 11128-11131. GARDINER, J. E., M. A. MCGANN,C. N. BERRIDGE, C. A. FULCHER, T. S . ZIMMERMAN & J. H. GRIFFIN.1984. Circulation 7 0 820. DAHLBACK, B. & B. HILDEBRAND. 1983. Biochem. J. 209: 857-863. HOSKINS,J., D. K. NORMAN,R. J. BECKMAN & G. L. LONG. 1987. Proc. Natl. Acad. Sci. USA 8 4 349-353. E. COHEN,M. SHAFFER,A. MAHR,B. DAHLBACK, J. LUNDWALL, A., W. DACKOWSKI, STENFLO& R. WYDRO.1986. Proc. Natl. Acad. Sci. USA 8 3 6716-6720. DAYHOFF, M. O., W. C. BARKER & L. T. HUNT. 1983. Methods Enzymol. 91: 524-545. QUE, B. G. & E. W. DAVIE.1986. Biochemistry 2 5 1525-1528. GRIFFITH,P., H. CHARBONNEAU, J. SHABANOWITZ, K. A. WALSH,D. F. HUNT& P. H. PETRA.In preparation.

4 199-4203.

32. 33. 34. 35. 36. 37.