Structural insights into lesion recognition and repair by the ... - Nature

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© 2002 Nature Publishing Group http://structbio.nature.com

Structural insights into lesion recognition and repair by the bacterial 8-oxoguanine DNA glycosylase MutM J. Christopher Fromme1 and Gregory L. Verdine1,2 Published online: 10 June 2002, doi:10.1038/nsb809

MutM is a bacterial 8-oxoguanine glycosylase responsible for initiating base-excision repair of oxidized guanine residues in DNA. Here we report five different crystal structures of MutM–DNA complexes that represent different steps of the repair reaction cascade catalyzed by the protein and also differ in the identity of the base opposite the lesion (the ‘estranged’ base). These structures reveal that the MutM active site performs the multiple steps of base-excision and 3′ and 5′ nicking with minimal rearrangement of the DNA backbone.

Reactive oxygen species (ROS) produced as by-products of aerobic respiration are among the most potent endogenous mutagens1. ROS attack DNA to generate a host of deleterious adducts, among which the most widely studied is 8-oxoguanine (oxoG or GO). Because of its tendency to mispair with adenine during replication, oxoG is a direct source of G-C-to-T-A transversion mutations. Nearly all organisms possess an elaborate defense system that confers resistance to the genotoxic effects of oxoG. In Escherichia coli, the ‘GO system’2–4 comprises three components. The first is MutT, an enzyme that sanitizes the nucleotide precursor pool by hydrolyzing 8-oxo-2′-deoxyguanosine-5′triphosphate (oxo-dGTP) to inorganic pyrophosphate and oxo-dGMP. Next, MutM (also known as 8-oxoguanine glycosylase or Fpg) effects the removal of oxoG lesion nucleosides from DNA via base-excision DNA repair chemistry. The third component, MutY, initiates the repair of misreplicated oxoG•A pairs in DNA by catalyzing excision of the adenine base. Bacteria possessing a defect in either the mutM or mutY genes alone show a modest increase in the rate of spontaneous mutagenesis, whereas mutM–mutY– strains show a >100-fold faster rate of mutagenesis relative to wild type, and mutM–mutY–mutT– triple-knockout strains display a 250-fold increase in mutation rate5. The GO system of eukaryotes is constructed along similar lines, having orthologs of MutT and MutY; however, MutM is replaced by a functionally analogous but structurally unrelated enzyme designated Ogg1 (refs 6,7). A major goal of research in this area is to explain the structural basis for substrate recognition and catalysis in the GO system. Toward this end, here we report five distinct structural snapshots of species that bracket the MutMmediated DNA repair pathway. Extensive biochemical analysis of the base-excision DNA repair process mediated by MutM has revealed that it entails a complex multistep reaction cascade. The sequence is initiated by nucleophilic substitution at the C1′ atom of the lesion, resulting in displacement of the oxoG base and covalent attachment of the N-terminal Pro residue8,9 (Pro 2) (Fig. 1). The aminal intermediate thus formed (2) rearranges to a ring-opened Schiff base (3), which undergoes sequential β- and δ-elimination reactions10, ultimately resulting in complete removal of the lesion-bearing 1

nucleoside from DNA but leaving behind the phosphates on the 5′ and 3′ side (4). The Schiff base (3) can be reductively intercepted in situ by sodium borohydride to generate the trapped complex 5 (ref. 11). MutM shows specificity not only for the lesion but also for the opposite DNA base. In terms of kcat/KM, the rank order of substrate preference is oxoG•T > oxoG•G > oxoG-C >> oxoG•A12. Presumably, there exists little selective pressure against the repair of oxoG•T and oxoG•G, because these are rarely encountered in vivo under ordinary circumstances. In contrast, effective exclusion of oxoG•A as a substrate is of critical biologic importance because it prevents mutagenic repair of misreplicated oxoG•A pairs to T-A. In addition to the oxoG lesion, MutM possesses the ability to repair several alternative substrates, most notably formamidopyrimidine (Fapy) lesions13–15 and 5-hydroxycytosine16. As the result of high-resolution crystallographic studies on human Ogg1, a great deal is now known about the structural basis for DNA damage recognition and removal by this enzyme17,18. Although MutM was discovered some nine years earlier than Ogg1 (refs 19,20), the structural biology of the bacterial system has lagged behind. A high-resolution structure of Thermus thermophilus MutM21 revealed that the overall fold of the enzyme is unrelated to that of Ogg1 but gave little insight into the details of substrate recognition and catalysis. Recently, Zharkov et al.22 reported the high-resolution structure of endonuclease VIII (endo VIII), a MutM homolog that repairs oxidized pyrimidines, borohydride-trapped to a DNA substrate. In the present report, we describe five different crystal structures of MutM from Bacillus stearothermophilus (B.st. MutM) in complex with lesion-containing DNA. These structures allow us to assign those residues most important for the cascade of reactions catalyzed by this enzyme and provide insight into how the enzyme recognizes various bases paired opposite oxoG. Overall structure of the complex The five structures reported here were solved by molecular replacement using the 1.9 Å crystal structure of unliganded Thermus thermophilus MutM21 as a search model and were further refined to 1.7–2.4 Å resolution (Table 1). The structures

Department of Molecular and Cellular Biology and 2Department of Chemistry and Chemical Biology, Harvard University, Cambridge, Massachusetts 02138, USA.

Correspondence should be addressed to G.L.V. email: [email protected] 544

nature structural biology • volume 9 number 7 • july 2002

articles O

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MutM

O O -O

O P

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DNA-O

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DNA-O

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Aminal intermediate, 2

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n d an atio β- limin δ-e

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End-product complex, 4

MutM

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Schiff base intermediate, 3

Na

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P O OH -O

+

N H MutM

O P

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Borohydride-trapped complex, 5

Fig. 1 Mechanistic scheme for the reaction cascade catalyzed by MutM. MutM is a bifunctional DNA glycosylase/β,δ-lyase. Excision of the oxidized guanine occurs via nucleophilic substitution at C1′ by the N-terminal proline (Pro 2). The aminal 2 thus formed rearranges to the Schiff base 3, which subsequently undergoes β- and δ- elimination, resulting in complete removal of the lesion nucleoside from DNA and formation of the end-product complex 4. The Schiff base can be reductively intercepted in situ by sodium borohydride to yield the trapped species 5. MutM also binds tightly to the reduced abasic site analog (rAb), furnishing complex 6. In this work, we present the structures of complexes 4 and 5 with the lesion opposite C. We also report structures of 6 with the lesion opposite C, G and T (rAb•C, rAb•G and rAb•T).

represent variations of MutM–DNA lesion recognition, with the differences being either in chemical composition and interactions within the active site or in the identity and recognition mode of the ‘estranged’ base directly opposite oxoG in the duplex. With the exception of these highly localized differences, the complexes have nearly identical overall structures (average r.m.s. deviations for Cα atoms = 0.18 Å), including the quaternary packing interactions in the crystal lattice (Table 1). To illustrate the general features of DNA recognition by MutM, we will first describe the 1.8 Å structure of MutM bound to DNA containing a reduced abasic site23 (6, Fig. 1) opposite cytosine (rAb•C). The N- and C-terminal domains of MutM straddle the DNA duplex, with the long axis of the protein oriented roughly orthogonal to the helical axis of the DNA (Fig. 2a). The protein is clasped onto the lesion-containing DNA strand, the backbone of which runs through a deep channel lined with basic residues at the midriff of the molecule where the two domains meet (Fig. 2c). MutM makes no direct contact with the backbone of the complementary strand. The DNA helix is sharply bent (∼75°) at the locus of the reduced abasic site and the estranged C (Fig. 2b) but possesses nearly ideal B-form structure in the flanking arms. These general features of DNA presentation to the glycosylase are strikingly similar to those observed with base-excision DNA repair proteins that have altogether different protein folds, most notably members of the UDG, HhH/GPD and Aag (super)families. Another conserved feature of DNA glycosylase–lesion complexes is the extrusion of the lesion from the DNA helix and its insertion into an extrahelical active site on the enzyme. Although the lesion base is not present in any of the structures reported here (despite our attempts to soak guanine analogs into the crystals), the conformations of the abasic nucleoside and its 5′ and 3′ phosphates project C1′ outward from the helix, consistent with extrahelical recognition and repair of the oxoG lesion. MutM splays open the minor groove and enforces the bend by inserting three side chains into the duplex: Met 77, Arg 112, and Phe 114. Phe 114 invades the DNA helix on the 5′ side of the estranged C, simultaneously making an edge–face interaction with the estranged C and a face–face interaction with its 5′ neighboring C (Figs 2d, 4a). Met 77 invades the helix right nature structural biology • volume 9 number 7 • july 2002

O

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NH

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P

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H 8N

P O

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DNA-O

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MutM

Reduced abasic site complex, 6

alongside Phe 114, stacking against the G residue on the 3′ side of the lesion and making van der Waals contacts with the lesion sugar at the C2′ and C3′ atoms. Arg 112 occupies the space left vacant by the extrahelical lesion, hydrogen bonding to the Watson-Crick face of the estranged C and π-stacking over the adenine base 5′ to the lesion (Figs 2d, 4a, vide infra). Arg 112 and Met 77 seem to play an especially important role in extrusion of the lesion base from the helix because these residues occupy the vacated space and direct the sugar moiety outward from the helix. The distorted DNA conformation observed in the MutM complexes is also enforced through an extensive array of hydrogen-bonding contacts to the phosphate moieties on the lesion-containing strand (Fig. 2d,e). Specifically, Lys 60, His 74, Tyr 242, Arg 264 and Lys 258 hydrogen bond to four successive phosphates, two of which lie on the 5′ side of the lesion and two on the 3′ side. Tyr 242, Lys 258 and Arg 264 are presented by a β-hairpin contained within a (Cys)4 zinc-finger motif in MutM, thus establishing a direct role for the zinc finger in DNA recognition24. Two residues, Lys 60 and Arg 264, are lodged between successive phosphates in the DNA and hydrogen bond to both; the former contact explains the essentiality of Lys 60 for MutM function25,26. An especially dense array of phosphate contacts is established at the site of the lesion (Fig. 3a). On the 5′ side, Tyr 242 contacts one of the two nonbridging oxygens, and Arg 264 contacts the other while also hydrogen-bonding to the bridging 5′ oxygen atom of the lesion. On the 3′ side, one nonbridging oxygen interacts with Lys 60, and the other participates in bifurcated hydrogen bonding to Arg 264. The contacts from Arg 264 to the phosphate groups 5′ and 3′ to the lesion nucleoside allow these phosphates to approach each other more closely than in normal B-form DNA (5.4 Å between the two closest nonbridging oxygens on neighboring phosphates compared to 6.7 Å in B-form DNA). These contacts, together with the strong intrinsic preference of phosphodiesters to adopt gauche conformations27, pinch the DNA backbone and force the sugar moiety of the lesion to jut outward from the DNA helix and into the heart of the active site. Such extensive backbone interactions at the site of the lesion have not been observed in other DNA glycosylase–lesion co-complexes, except in that of the MutM homolog endonucle545

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** * * MPELPEVETIRRTLLPLIVGKTIEDVRIFWPNIIRHPRDSEAFAARMIGQTVRGLERRGK 60 MPELPEVETTRRRLRPLVLGQTLRQVVHRDPARYRNTALAE-------GRRILEVDRRGK 53 MPELPEVETSRRGIEPHLVGATILHAVVRN-GRLRWPVSEEIYRLS--DQPVLSVQRRAK 57

B.st. T.th. E.co.

* * * * FLKFLLDR-DALISHLRMEGRYAVASALEPLEPHTHVVFCFTDGSELRYRDVRKFGTMHV 119 FLLFALEGGVELVAHLGMTG----GFRLEP-TPHTRAALVL-EGRTLYFHDPRRFGRLFG 107 YLLLELPEGW-IIIHLGMSGSLRILPEELPPEKHDHVDLVMSNGKVLRYTDPRRFGAWLW 116

B.st. T.th. E.co.

YAKEEADRRPPLAELGPEPLSPAFSPAVLAERAVKTKRSVKALLLDQTVVAGFGNIYVDE 179 VRRGDYREIPLLLRLGPEPLSEAFAFPGFFRGLKESARPLKALLLDQRLAAGVGNIYADE 167 TKELEGHN--VLTHLGPEPLSDDFNGEYLHQKCAKKKTAIKPWLMDNKLVVGVGNIYASE 174

B.st. T.th. E.co.

SLFRAGILPGRPAASLSSKEIERLHEEMVATIGEAVMKGGSTVR--TYVNTQGEAGTFQH 237 ALFRARLSPFRPARSLTEEEARRLYRALREVLAEAVELGGSTLSDQSYRQPDGLPGGFQT 227 SLFAAGIHPDRLASSLSLAECELLARVIKAVLLRSIEQGGTTLKD--FLQSDGKPGYFAQ 232

B.st. T.th. E.co.

* * * * * * * HLYVYGRQGNPCKRCGTPIEKTVVAGRGTHYCPRCQR--- 274 RHAVYGREGLPCPACGRPVERRVVAGRGTHFCPTCQGEGP 267 ELQVYGRKGEPCRVCGTPIVATKHAQRATFYCRQCQK--- 269

ase VIII22, suggesting perhaps that proteins of the MutM/endo VIII structural class stabilize the extrahelical conformation more through contacts to the backbone of the lesion than to its base moiety (vide infra). Recognition of the estranged base The prototypical substrates of MutM are oxidized versions of guanine paired opposite cytosine. However, E. coli MutM shows considerable flexibility with respect to the identity of the estranged base, repairing oxoG opposite thymine (oxoG•T) or guanine (oxoG•G) with greater catalytic efficiency (kcat/KM) than oxoG-C12. This is in contrast to Ogg1, which shows an overriding preference for oxoG-C versus any other oxoG•N pair6,7. To explain the mode of estranged base recognition by MutM, we crystallized and determined the structures of two ‘mismatched’ recognition complexes: the reduced abasic site opposite thymine (rAb•T, refined to 2.2 Å) and the reduced abasic site paired opposite guanine (rAb•G, refined to 2.0 Å) (Table 1). Not sur546

Fig. 2 Overall structure of the MutM–DNA complex (rAb•C). a, Ribbon diagram of the complex, with α-helices in red, β-strands in blue, nonrepetitive elements in gray and DNA in gold; the zinc atom is shown as a pink sphere. The estranged cytosine is represented by a magenta bar. b, Same as in (a) but viewed from a different perspective. c, Electrostatic surface (GRASP)39 representation in the region near the MutM–DNA interface. Red represents negatively charged surfaces; blue, positively charged surfaces. The estranged cytosine is in magenta. The DNA has been truncated in this view for purposes of visual clarity. d, MutM–DNA interface. DNA carbon atoms are gold, the MutM backbone is light gray, side chain carbon atoms are either green or cyan, the lesion nucleoside is dark gray and the estranged base is magenta; this coloring scheme is retained throughout. e, Schematic of MutM contacts to the phosphate backbone of the lesion-containing DNA strand. Dotted lines denote hydrogen bonds. f, MutM amino acid sequence alignment. Conserved residues are highlighted in red. Asterisks above residues denote those important for DNA recognition and catalysis, and are color coded according to the same scheme used for all figures. Yellow asterisks denote the residues involved in zinc coordination. All residues explicitly mentioned in the body of the text, excluding Lys 258, are conserved among mutM genes from different organisms. B.st. corresponds to Bacillus stearothermophilus; T.th., Thermus thermophilus; and E.co. Escherichia coli.

prisingly, the overall structures of the mismatched complexes are nearly identical to that of the prototypical rAb•C recognition complex, but differences are apparent in the vicinity of the estranged base. A single amino acid, Arg 112, makes a specific interaction with the estranged base. With cytosine in the estranged position, the base adopts the usual anti glycosidic rotamer (Fig. 4a), thereby presenting its O2 and N3 hydrogen bond acceptor atoms for bidentate interaction with NεH and NηH of the Arg 112 side chain guanidinium group. The estranged guanine, in contrast, adopts the syn glycosidic rotamer, presenting its Hoogsteen face (O6 and N7) for bidentate hydrogen bonding with the Arg 112 NεH and NηH (Fig. 4b). The mode of interaction with an estranged thymine is less straightforward than with C or G. Arg 112 clearly approaches the Watson-Crick face of the T (anti conformer), but the functionality on the T is not perfectly matched with that on Arg 112; hence, the two partners are farther removed from one another than in the rAb•C and rAb•G nature structural biology • volume 9 number 7 • july 2002

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structures (Fig. 4c). The O2 carbonyl of the estranged T lies within hydrogen bonding distance of Nε, but the N1 of T is retracted from Nη of Arg 112 to avoid a counterproductive NH-NH clash between the two. Presumably owing to the lack of a suitable T–Arg interaction mode, the electron density maps indicate a significant static disorder in the estranged T, whereas the estranged C and G appear to be well ordered. The efficiency with which MutM processes any substrate is strongly influenced by several factors: (i) the competence of the enzyme–substrate complex for catalysis (ii) the magnitude of the favorable interactions between the lesion-containing duplex and the enzyme upon formation of a specific recognition complex and (iii) the stability of the lesion-containing substrate duplex and, hence, the energetic cost of base extrusion from the helix. Although disentangling these three factors on the basis of crystal structures alone is not possible, the present data, together with previous biochemical results, allow some proposals concerning estranged base selectivity to be advanced. Implicit in our analysis nature structural biology • volume 9 number 7 • july 2002

Fig. 3 Comparison of active sites from three different stages of catalysis. a, Active site structure of the rAb•C recognition complex 6. The density corresponding to the C1′ atom and its hydroxyl is weak, presumably owing to several rotamers about the C2′-C3′ bond. b, Superposition of the final rAb•C complex model and its final 1.8 Å σA37-weighted Fo – Fc electron density map, calculated using simulated-annealing omit phases and contoured at 3 σ. Note the disorder at C1′ and O1′, and the helixcapping interaction of Glu 3. The extra density near N of Pro 2 may be due to water, buffer or an interaction with O1′. c, Active site of the borohydride-trapped complex 5. Note the covalent bond between N of Pro 2 and C1′ of the lesion nucleoside (magenta) and the location of the ordered water (pink). Note also the absence of the oxoG base. d, The final 1.7 Å σA-weighted Fo – Fc density map of the borohydride-trapped complex, calculated and contoured as in (b) and superimposed on the structure from (c). e, Structure of the active site region of the end-product complex 4. The sugar has diffused out of the active site leaving behind its 5′ and 3′ phosphate groups. f, The end-product complex 2.4 Å σA-weighted Fo – Fc electron density map calculated using simulatedannealing omit phases, contoured at 2 σ and superimposed on the structure from (e).

is the assumption that B.st. MutM has a similar substrate preference as E. coli MutM; this is not unreasonable given the high sequence identity (40%) and conservation of key residues involved in DNA recognition and catalysis. The forward rate constant (kcat) values of MutM for oxoG•T and oxoG•G are roughly equal but are approximately five-fold higher than oxoG-C12. The present structures suggest that the oxoG-C substrate makes the most productive interactions with MutM, the oxoG•G–MutM complex is of intermediate stability because of its syn glycosidic conformation and the oxoG•T–MutM complex is least stable. In contrast, an oxoG-C DNA duplex is the most costly of the three to disrupt, oxoG•T is intermediate and oxoG•G least costly28. These factors amount to make oxoG•T and oxoG•G roughly equivalent as substrates in terms of kcat/KM but more efficient than oxoG-C by about an order of magnitude. From a biologic standpoint, the key question concerns the mechanism by which MutM excludes oxoG•A. Biochemical studies have revealed that the selectivity against oxoG•A results 547


articles mainly from relatively poor binding of the enzyme to this substrate12. We envision that this poor binding results in part from ineffective recognition of an estranged A by Arg 112. The Watson-Crick and Hoogsteen faces of A in the anti or syn glycosidic rotamers, respectively, are chemically mismatched with the hydrogen-bonding functionality on Arg. Furthermore, the unusually high stability of the oxoG•A base pair in DNA28 provides an additional penalty weighting against formation of a stable recognition complex. Active site architecture and catalysis MutM catalyzes conversion of its substrate through multiple reaction steps, during which the base is expelled and the linkages of the 3′ and 5′ carbons to the respective phosphates are cleaved (Fig. 1). It remains of great interest to elucidate the mechanism by which MutM carries out this cascade of chemical reactions using a single active site. To gain further insight into this aspect of MutM function, we have solved the 1.7 Å structure (Table 1) of a borohydride-trapped complex in which Pro 2 is covalently

linked to the C1′ atom of the substrate 2′-deoxyribose sugar (5, Fig. 1). This trapped complex differs from the authentic Schiff base intermediate 3 only by the relatively conservative replacement of a C=N double bond with a C–N single bond. We have also solved the 2.4 Å structure (Table 1) of an end-product complex formed between MutM and a fully processed substrate (4, Fig. 1). The differences between the structures of complexes 4, 5 and 6 are all localized to the active site region. The active site of MutM (Fig. 3) lies at the interface between the two domains of the protein and is composed of residues from both. Pro 2, the residue responsible for initiating the repair reaction cascade via nucleophilic attack29, is located at the N-terminal end of a long α-helix (α-A), the macrodipole of which interacts favorably with the negative charge of the DNA backbone. Pro 2 appears to be in nearly perfect position for attack on the C1′ atom of the deoxyribose ring of the lesion (Fig. 3a,b). This precise positioning is determined through both the local torsional preferences of Pro 2 and hydrogen bonding from the Glu 3 amide NH to the side chain of Glu 6. The conformation of

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Fig. 4 Recognition of the estranged base by MutM. a, Recognition in the rAb•C complex. b, Recognition in the rAb•G complex. c, Recognition in the rAb•T complex. Dashes indicate hydrogen bonds, and red circles denote a van der Waals interaction. d–f, Structural formulae schematics of the mode of recognition seen in (a–c), respectively.

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articles Table 1 Data collection and model statistics


Complex

Borohydridetrapped intermediate 1L1Z

End-product complex

rAb•T rAb•G recognition complex recognition complex

PDB entry

Reduced abasic (rAb•C) recognition complex 1L1T

1L2B

1L2C

1L2D

Data collection Resolution (Å) Unique reflections Redundancy Completeness (%)1 Rmerge1,2 1

50–1.8 41,946 6.0 99.6 (96.4) 0.067 (0.53) 26.7 (2.0)

30–1.7 47,000 5.3 96.7 (76.9) 0.067 (0.417) 25.2 (2.2)

50–2.4 12,648 2.0 84.6 (82.5) 0.101 (0.445) 8.7 (1.9)

50–2.2 22,900 4.5 98.3 (94.6) 0.135 (0.469) 10.9 (2.9)

50–2.0 30,349 5.6 99.2 (94.0) 0.127 (0.470) 14.0 (2.7)

1.7 22.1 (22.6) 24.6 (25.0) 28.7

2.4 20.9 (22.4) 26.5 (27.4) 41.7

2.2 21.3 (21.4) 24.7 (28.6) 43.0

2.0 21.7 (21.2) 25.1 (25.4) 35.8

0.006 1.22 22.8

0.007 1.25 23.0

0.006 1.18 22.4

0.006 1.16 22.4

91.9 7.7 0.5 2,462 234

89.1 10.4 0.5 2,464 124

92.6 6.9 0.5 2,438 141

93.1 6.5 0.5 2,441 181

Refinement and model statistics Resolution (Å) 1.8 Rcryst1,3 21.4 (22.2) Rfree1,3 23.8 (26.0) Mean B-value, all atoms (Å2) 30.8 R.m.s. deviation from ideality Bond lengths (Å) 0.006 Bond angles (°) 1.20 Dihedral angles (°) 22.4 Ramachandran plot (%) Most favored 91.2 Additionally allowed 8.3 Generously allowed4 0.5 Protein and DNA atoms 2,439 Water 231

Values in parentheses refer to the highest resolution bin. Rmerge = Σhkl |I(hkl) – | / Σhkl . 3R cryst = Σhkl |Fo(hkl) – Fc(hkl)| / Σhkl |Fo (hkl)|. Rfree was calculated based on 10% of the total data omitted during structure refinement. 4One residue (Arg 76) fell in this region of φ-ψ space in all five structures. It was inspected with the final σA37-weighted 2F – F map and determined o c to be appropriately modeled. 1 2

Glu 3 is also highly restricted. The Glu 3 side chain carboxylate acts as an N-terminal cap for helix α-E, the macrodipole of which also interacts favorably with the DNA backbone. The amide carbonyl of Glu 3, in turn, initiates helix α-A. In the reduced abasic and borohydride-trapped complexes (6 and 5, respectively), Glu 3 also hydrogen bonds with the O4′ hydroxyl of the lesion ribose. The positioning of the Glu 3 N-capping interaction with helix α-E in the active site of the MutM co-crystal structures bears a striking similarity to the Asp 268 N-capping interaction with helix α-M in the active site of hOgg1 (refs 17,18). This local structural similarity in the core of the active site was not expected because MutM and hOgg1 possess completely unrelated folds and use different residues as the catalytic nucleophile (N-terminal Pro versus internal Lys, respectively). The active site Asp residue is the only invariant active site residue in the large HhH-GPD superfamily of DNA glycosylases to which hOGG1 belongs7 (but not MutM). This active site Asp is essential for catalysis of base excision and is observed to act as an N-cap for the α-M helix in every structure of an HhH-GPD glycosylase reported so far17,30–32. The active site Asp residue of HhH-GPD glycosylases has been proposed to stabilize the oxocarbenium ion character in the transition state for base excision18. In analogy, we propose that Glu 3 of MutM serves an analogous role in oxocarbenium ion stabilization. Electron density maps for the borohydride-trapped complex (Fig. 3d) show continuous electron density between Pro 2 and the C1′ atom of the substrate 2′-deoxyribose moiety; the distances between these two atoms and their geometries are also nature structural biology • volume 9 number 7 • july 2002

consistent with the existence of a covalent bond between the enzyme and substrate. The excised oxoG base diffuses away, and our attempts at soaking or co-crystallizing with various guanine analogs failed to introduce a base into the active site, implying that the enzyme does not bind tightly to the excised product base. This is in contrast to hOgg1, which holds onto the displaced oxoG residue long after the base excision step, and even after borohydride trapping (J.C.F., S.D. Bruner and G.L.V., unpublished results). The conformation of the DNA backbone is remarkably similar in complexes 5 and 6, despite the observation that one bears a covalent linkage to the enzyme and the other does not (compare Fig. 3a and c). This observation is consistent with the notion that MutM exerts tight control over the backbone conformation through an extensive array of intermolecular contacts. In comparison with the active site of the rAb lesion, that of the borohydride-trapped complex has a slight change in the orientation of Pro 2, resulting from rotation about the Cα-Cβ bond (χ1). The active site of the end-product complex 4 is completely devoid of electron density attributable to either the base or sugar moiety of oxoG, indicating complete excision of the lesion from DNA (Fig. 3e,f). The phosphates formerly attached to the 5′ and 3′ positions of the oxoG are clearly visible, as expected. Only subtle shifts in the conformation of the DNA backbone and active site residues are observed in 4 relative to those in 5 and 6. For example, Arg 264 has become more flexible and the conformation of the 3′ phosphate group has changed slightly, acquiring an extra hydrogen bond to Lys 60. 549


articles MutM catalyzes proton abstraction from both the C2′ (β-elimination step) and C4′ (δ-elimination step) atoms. These steps are generally considered to be chemically demanding, leading to the expectation that residues of MutM capable of acting as general base catalysts would be poised for catalysis near C2′ and C4′. Although Glu 3 is in the general vicinity of both C2′ and C4′, this residue has been ruled out as the general base in the case of the MutM homolog endonuclease VIII22. No other residue on the enzyme appears capable of performing the proton abstractions, absent a major rearrangement of the active site, which is unlikely based on the conservation of active site structure in the product complex 4. In the structure of the borohydride-trapped complex 5, we observed an ordered water (Fig. 3c,d) positioned 3.5 Å away from the C2′ atom, with perfect alignment to remove the proS proton from this carbon. Thus, ordered water molecules may act as specific base catalysts to perform the proton abstraction steps at the C2′ and C4′ atoms. Assessment of this proposal will require further biochemical and structural studies, including determination of the stereochemistry of proton abstraction. Both elimination steps are undoubtedly facilitated by the extensive contacts between the 5′ and 3′ phosphate oxygens and residues Arg 264, Lys 60 and Tyr 242. Such interactions could accommodate the buildup of negative charge on the phosphates that accompanies elimination. The extent of similarity in structure of the complexes 4, 5 and 6 is striking considering that these three structures span different stages of base-excision DNA repair by MutM. The active site of MutM thus appears to have evolved to manage the transition from reaction intermediates to the final product with economy of motion.

is the structure of the protein affected? In the case of hOGG1, comparison of the lesion-bound17,18 and unbound structures33 revealed that the overall fold of the protein is virtually unaffected by binding to a lesion, with the only significant changes occurring in the immediate vicinity of the active site. An analogous comparison in the case of MutM is complicated by the fact that sequences of the B. stearothermophilus (present work, liganded) and T. thermophilus21 (unliganded) are only 43% identical. However, despite this caveat, the overall fold and relative disposition of the two domains clearly are similar (r.m.s. deviation for Cα atoms = 1.2 Å). The most significant difference is seen in the ∼10-amino acid segment proposed here to be involved in lesion base recognition; this segment is disordered in the DNA-bound structure but ordered in the unbound structure even though neither structure contains an oxoG base. The tip of the conserved loop containing Arg 264, the key residue that contacts the phosphates on either side of the lesion (Fig. 3), draws inward toward DNA upon binding. Other than these two changes in the protein backbone, the remaining differences involve side chain rotations at His 74, Met 77 and Phe 114. The disposition of Arg 112, the residue that hydrogen bonds directly to the estranged base, is unchanged in the lesion-bound versus unliganded structures. Note added in proof: After this paper was submitted, the structure of E. coli MutM borohydride-trapped to DNA was reported40. Methods Cloning, overexpression and purification. The mutm gene was identified in the B. stearothermophilus genome by a BLAST search (http://www.genome.ou.edu/bstearo.html). Colony PCR was performed on B. stearothermophilus cells using the primers 5′-GGCTAACATATGCCGGAATTGCCGGAGGTGGAAACG-3′ and 5′-TTATT ATGCGGCCGCTTAGCGCTGGCAGCGCGGGCAATAATGCG-3′, and the resulting product was cloned into the pET24 (Novagen) expression vector using NdeI and NotI, resulting in an ORF encoding the natural sequence of B.st. MutM with no modifications. The plasmid was transformed into BL21(DE3)pLysS cells, which were allowed to grow to an OD600 of 0.5 at 37 °C. Overexpression was induced by the addition of 0.5 mM IPTG, followed by the addition of 50 µM ZnCl2 and shaking of the culture at 30 °C for 4 h. Cells were harvested by centrifugation. Pellets were resuspended in 50 mM NaPO4, pH 8.0, 500 mM NaCl and 0.1% βME (∼3 ml buffer g–1 of cell pellet), snap frozen in liquid nitrogen and stored at –80 °C. Cells were thawed in cold water and lysed by French press after the addition of phenylmethylsulfonyl flouride (PMSF) to 1 mM concentration. DNase and RNase were added to the lysates at a final concentration of 0.1 mg ml–1 for each, and lysates were clarified by centrifugation at 20,000g for 25 min. Clarified lysates were diluted 1:5 in Buffer A (20 mM Tris, pH 7.4, and 0.1% βME) and loaded onto a 20 ml SFF column. A linear gradient of Buffer A to Buffer B (Buffer A + 1 M NaCl) was passed over the column, and fractions determined by SDS-PAGE analysis to contain MutM were pooled. NaCl was added to a final concentration of ∼1 M, and the pool was concentrated using an Amicon YM-10 centrifugal concentrator and run on a Superdex 75 gel filtration column equilibrated with 10 mM Tris, pH 7.4, 1 M NaCl and 5 mM βME. The peak fractions containing MutM were pooled, concentrated to 10 mg ml–1 and stored at 4 °C under argon. In the absence of DNA, high concentrations of NaCl were required to maintain the solubility of the protein.

Lesion base recognition Although our structures do not include an oxidized lesion base, it is possible to infer the location of the base-recognition pocket from the structures presented here. The base recognition pocket appears to be bordered on two sides by the A and E helices, and residues at the N-terminal ends of these helices may play a role in base recognition. One face of the base recognition pocket is completely open in our structure because of the lack of ordered structure in a large loop consisting of residues 221–234 (dotted line, Fig. 2a,b). Remarkably, this loop is ordered in the MutM structure in the absence of DNA21 and seals the active site precisely over the region in which the lesion base would be located. We propose that this loop becomes ordered in the presence of a lesion base and forms a key component of the lesion-recognition pocket. Only three conserved residues are located in this putative lesion-recognition loop: Thr 221, Gly 230 and Gly 233. These are obvious candidates for residues that either directly recognize the base or serve a crucial structural role. There is also conservation of an aromatic side chain functionality at position 235, and it is tempting to speculate that this side chain stacks against the lesion base analogously to Phe 319 of hOgg1 (refs 17,18). The observation that this loop is flexible has interesting implications for the activity of MutM. Perhaps this flexibility explains why MutM fails to hold onto its lesion base following excision, suggesting that the interactions with the free base are insufficient to pay the entropic cost of ordering the lesion-recognition loop. The structural plasticity of this loop may be what allows MutM DNA preparation, complex formation and borohydride trapping. The DNA used for crystallization was either purchased to repair such a diverse array of lesions16. MutM structural changes accompanying DNA binding Formation of a specific recognition complex with DNA bound to MutM results in drastic remodeling of duplex structure. How 550

from Operon Technologies or synthesized on an ABI 392 DNA synthesizer using standard reagents, except for the reduced abasicsite phosphoramidite synthesized in our lab (gift of H.M. Nash) and the 8-oxoguanosine phosphoramidite purchased from Glen Research. The DNA sequence used for crystallization was



articles 5′-TGCGTCCAXGTCTACC-3′ annealed to 5′-AGGTAGACYTGGACGC- 3′, where ‘X’ was either the reduced abasic-site (all three recognition complexes) or 8-oxoguanosine (borohydride-trapped and end-product complexes), and ‘Y’ was C for the rAb•C, borohydride-trapped and end-product complexes, and G for rAb•G or T for rAb•T complexes. The growth of crystals occurred using several different lengths of DNA, ranging from a 13mer to a 17mer. The 15mer duplex described above was used for the studies presented in this work. All DNA was PAGE purified on 20% (w/v) polyacrylamide, 8 M Urea and 1× TBE gels. The DNA duplexes were stored in 10 mM Tris, pH 8.0, at 4 °C. DNA–protein complexes were prepared by adding 1.5 molar equivalents of DNA to MutM at 10 mg ml–1 in 1 M NaCl, then adding sufficient 10 mM Tris, pH 7.4, to dilute the [NaCl] to 50 mM and concentrating the complex down to a final MutM concentration of 7.5 mg ml–1. The borohydride-trapping reaction was prepared using 2.5 molar equivalents of a DNA solution containing NaBH4 and glycerol to final concentrations of 100 mM and 5% (v/v), respectively. Dilution with 10 mM Tris, pH 7.4, was carried out so that the concentration of these two components did not change. The trapping reaction was monitored by SDS-PAGE and reached ∼60% completion with respect to protein. The trapped complex was purified using a 1 ml MonoQ column and a linear gradient of Buffers A and B described above; buffer exchanged into 10 mM Tris, pH 7.4, 5 mM βME and 50 mM NaCl; and concentrated to a final MutM concentration of ∼8 mg ml–1. The trapped complex was judged to be >99% pure by SDS-PAGE analysis. Crystallization, data collection and structure solution. All complexes were crystallized by the 1:1 vapor-diffusion method using 13–18% (w/v) PEG 8000, 100 mM sodium cacodylate, pH 7.5, 50 mM Mg(CH3COO)2 and 0.1 % βME. Crystals appeared within several days and grew to final dimensions of 500 × 100 × 75 microns within two weeks. The crystals belonged to the orthorhombic space group P212121, with unit cell dimensions of a = 44 Å, b = 94 Å and c = 103 Å. Crystals were transferred to a solution of 18% (w/v) PEG 8000, 100 mM sodium cacodylate, pH 7.5, 50 mM Mg(CH3COO)2, 0.1% (v/v) βME and 25% (v/v) glycerol for cryoprotection before freezing in liquid nitrogen for data collection. All data were collected at 100 K at the A1 beamline of CHESS (Cornell High-Energy Synchrotron Source). Data was processed using DENZO/SCALEPACK34. Data collection statistics are summarized in Table 1. The structure of the recognition complex (rAb•C) was solved by molecular replacement using all atoms present in one monomer of T. thermophilus MutM (PDB entry 1EE8 (ref. 21), 43% identity) as a search model in cross-rotation and translation function searches performed using CNS 1.0 (ref. 35). The initial model gave an Rcryst of 51.8% and Rfree36 of 52.0% after rigid-body refinement. Simulated annealing and grouped B-factor refinement of the search model using data to 2.5 Å generated a model with Rcryst of


40.5% and Rfree of 44.1%. A σA-weighted37 2Fo – Fc map calculated using this model and all reflections >2 σ was of excellent quality, allowing facile interpretation of differences between the search model and the actual structure. Our attempts at density modification failed to improve the map, probably owing to the initially unobservable electron density of the bound DNA and our inability to construct a satisfactory solvent mask around the complex due to the unknown orientation of the DNA in the crystal. Subsequent rounds of model building of the protein, performed in Quanta98 (Molecular Simulations), and refinement using CNS35 resulted in σA-weighted 2Fo – Fc maps in which the DNA became clearly visible. After all observable protein and DNA atoms were built into the model, water molecules were automatically added using CNS and manually inspected to confirm their location. The structures of the borohydride-trapped complex, end-product complex and two mismatch recognition complexes (rAb•T and rAb•G) were determined using differenceFourier techniques based on the recognition complex structure. Additional residues were built into these structures as necessary, and all structures were refined by subsequent rounds of positional refinement, torsion-angle-simulated annealing, energy minimization and individual B-factor refinement while monitoring Rfree. In all models, several surface residue side chains were truncated at Cα, Cβ or Cγ if electron density was not visible for the entire side chain. The final model consists of 12 base pairs of DNA and the full-length protein, excluding residues 224–232 in the end-product structure, 223–232 in the borohydride-trapped structure and 221–234 in all three recognition complex structures. Model statistics and geometries (PROCHECK)38 for all of the structures are presented in Table 1. Coordinates. The coordinates for the structures have been deposited in the Protein Data Bank (accession codes 1L1T (rAb•C), 1L1Z (borohydride-trapped complex), 1L2B (end-product complex), 1L2C (rAb•T) and 1L2D(rAb•G)).

Acknowledgments We are grateful to B. Roe and S. Lewis of the OU Advanced Center for Genome Technology for their help with B. stearothermophilus. The entire staff of MacCHESS, especially C. Heaton and B. Miller, provided valuable assistance with data collection and processing. We thank M. Spong for assistance with the crystallization screens, and members of the Verdine, Harrison and Wiley labs for thoughtful discussions. J.C.F is funded by an NSF predoctoral fellowship and an NIH training grant. Competing interests statement The authors declare that they have no competing financial interests. Received 5 March, 2002; accepted 6 May, 2002.

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