A plasmid-encoded dihydrofolate reductase from trimethoprim-resistant bacteria has a novel D2-symmetric active site

Narayana, Narendra; Matthews, David A.; Howell, Elizabeth E.; Xuong, Nguyen‐Huu

doi:10.1038/nsb1195-1018

Cited by 78 publications

(213 citation statements)

References 35 publications

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“…In these systems, amide group interactions have been proposed to play an important role in N5 protonation. The absence of analogous interactions in the Type II DHFR ternary complex is probably an important reason for its relatively low catalytic activity (5). In addition to the limited role that the pteridine amide can play in N5 protonation, there is no obvious protonation mechanism/pathway that can be identified in the ternary complex studied here.…”

Section: Discussionmentioning

confidence: 65%

“…Thus, the interaction of the N3-O4 amide of the pteridine ring with Ile68 makes a critical contribution to DHF recognition and to reactivity. Interestingly, the pteridine ring of DHF is laterally displaced relative to its position in the previously reported folate binary complex (5). This leads to hydrogen bond distances of 2.88 Å and 2.98 Å between N3-Ile68 O and O4-Ile68 N in the present structure, compared with corresponding distances of 4.75 and 3.07 Å in the DHFR•(folate) 2 structure (5).…”

Section: Substrate/cofactor Recognitionmentioning

confidence: 56%

“…organized into a toroidal structure with a central pore (5). Structural studies indicate that both the substrate (5) and cofactor (6)(7)(8) bind in this central pore, which functions as the active site.…”

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confidence: 99%

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Crystal Structure of a Type II Dihydrofolate Reductase Catalytic Ternary Complex

et al. 2007

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Type II dihydrofolate reductase (DHFR) is a plasmid-encoded enzyme that confers resistance to bacterial DHFR-targeted antifolate drugs. It forms a symmetric homotetramer with a central pore which functions as the active site. Its unusual structure, which results in a promiscuous binding surface that accommodates either the Dihydrofolate (DHF) substrate or the NADPH cofactor, has constituted a significant limitation to efforts to understand its substrate specificity and reaction mechanism. We describe here the first structure of a ternary R67 DHFR•DHF•NADP + catalytic complex, resolved to1.26 Å. This structure provides the first clear picture of how this enzyme, which lacks the active site carboxyl residue that is ubiquitous in Type I DHFRs, is able to function. In the catalytic complex, the polar backbone atoms of two symmetry-related I68 residues provide recognition motifs that interact with the carboxamide on the nicotinamide ring, and the N3-O4 amide function on the pteridine. This set of interactions orients the aromatic rings of substrate and cofactor in a relative endo geometry in which the reactive centers are held in close proximity. Additionally, a central, hydrogen-bonded network consisting of two pairs of Y69-Q67-Q67′-Y69′ residues provides an unusually tight interface, which appears to serve as a "molecular clamp" holding the substrates in place in an orientation conducive to hydride transfer. In addition to providing the first clear insight regarding how this extremely unusual enzyme is able to function, the structure of the ternary complex provides general insights into how a mutationallychallenged enzyme, i.e., an enzyme whose evolution is restricted to four-residues-at-a-time active site mutations, overcomes this fundamental limitation. KeywordsR67 DHFR; Dihydrofolate Reductase; X-ray crystallography; ternary complex; Type II DHFR Antifolate drug therapy plays a critical role in the treatment of pathogenic and neoplastic diseases. The evolution of a plasmid-encoded, Type II dihydrofolate reductase (DHFR) provides one mechanism for bacterial evasion of drugs such as trimethoprim that target the bacterial dihydrofolate reductase enzyme (1-4). Type II DHFR is an extremely unusual enzyme that exhibits no apparent structural or evolutionary relationship with the type I (chromosomal) enzyme. It is one of the smallest enzymes known to self-assemble into an active quaternary structure, forming a homotetramer consisting of four 78-residue peptides * To whom correspondence should be addressed. This type of active site structure also creates substantial evolutionary and mutational challenges to the enzyme. Since each mutation will alter four active site residues at a time, most of the evolutionary pressure that would normally optimize enzyme function is compromised by the need to balance the effects of substitutions at all four symmetry-related sites. For example, a residue substitution on one monomer, which might promote folate N5 protonation, may also interfere with NADPH binding when it is prese...

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Section: Discussionmentioning

confidence: 65%

Section: Substrate/cofactor Recognitionmentioning

confidence: 56%

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Crystal Structure of a Type II Dihydrofolate Reductase Catalytic Ternary Complex

et al. 2007

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“…The unusual ͞ angles are not an absolute requirement for the SH3 domain fold because they are not present in other SH3-like proteins unrelated in sequence (38)(39)(40). In SH3 domains, the Gly-48 position is close to Pro-51, which is also almost universally conserved and is crucial for peptide-binding activity.…”

Section: Discussionmentioning

confidence: 99%

Dramatic acceleration of protein folding by stabilization of a nonnative backbone conformation

Nardo

Korzhnev

Stogios

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

100

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Through a mutagenic investigation of Gly-48, a highly conserved position in the Src homology 3 domain, we have discovered a series of amino acid substitutions that are highly destabilizing, yet dramatically accelerate protein folding, some up to 10-fold compared with the wild-type rate. The unique folding properties of these mutants allowed for accurate measurement of their folding and unfolding rates in water with no denaturant by using an NMR spin relaxation dispersion technique. A strong correlation was found between ␤-sheet propensity and the folding rates of the Gly-48 mutants, even though Gly-48 lies in an unusual non-␤-strand backbone conformation in the native state. This finding indicates that the accelerated folding rates of the Gly-48 mutants are the result of stabilization of a nonnative ␤-strand conformation in the transition-state structure at this position, thus providing the first, to our knowledge, experimentally elucidated example of a mechanism by which folding can occur fastest through a nonnative conformation. We also demonstrate that residues that are most stabilizing in the transition-state structure are most destabilizing in the native state, and also cause the greatest reductions in in vitro functional activity. These data indicate that the unusual native conformation of the Gly-48 position is important for function, and that evolutionary selection for function can result in a domain that folds at a rate far below the maximum possible. T he energetics involved as proteins progress from a broad ensemble of unfolded conformations to a single native structure are still not thoroughly understood. A widely used approach to study folding is the protein engineering method, which measures the thermodynamic and kinetic effects of single amino acid substitutions (usually with Ala) at many different sites in a protein as a means to define the most structured regions of the folding transition state (1, 2). Mapping the transition state identifies regions that fold more quickly than others, and such knowledge has led to important theoretical advances in our understanding of the folding process (3, 4). Although this method provides a general picture of folding pathways, it does not define the mechanisms by which individual residues can facilitate or retard the folding process. Our approach to study these mechanisms is to investigate the effects of multiple amino acid substitutions at single positions (5-7). In the present study, this approach is used to investigate the role in folding and function of position 48 of the Src homology 3 (SH3) domain, which is one of most conserved positions in this domain (8).The SH3 domain is well suited for protein-folding studies due to its small size and amenability to biophysical analysis (9-11). Composed of two three-stranded ␤-sheets packed orthogonally against one another (Fig. 1A), SH3 domains function as proteinprotein interaction modules in a wide variety of eukaryotic proteins. The structure of the SH3 domain transition state has been extensively characterized...

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“…1 (5). The symmetry of the active site results in overlapping binding sites for substrate, DHF, and cofactor, NADPH.…”

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A Balancing Act between Net Uptake of Water during Dihydrofolate Binding and Net Release of Water upon NADPH Binding in R67 Dihydrofolate Reductase

Chopra

Dooling

Horner

et al. 2008

Journal of Biological Chemistry

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R67 dihydrofolate reductase (DHFR) catalyzes the reduction of dihydrofolate (DHF) to tetrahydrofolate using NADPH as a cofactor. This enzyme is a homotetramer possessing 222 symmetry, and a single active site pore traverses the length of the protein. A promiscuous binding surface can accommodate either DHF or NADPH, thus two nonproductive complexes can form (2NADPH or 2DHF) as well as a productive complex (NADPH⅐DHF). The role of water in binding was monitored using a number of different osmolytes. From isothermal titration calorimetry (ITC) studies, binding of NADPH is accompanied by the net release of 38 water molecules. In contrast, from both steady state kinetics and ITC studies, binding of DHF is accompanied by the net uptake of water. Although different osmolytes have similar effects on NADPH binding, variable results are observed when DHF binding is probed. Sensitivity to water activity can also be probed by an in vivo selection using the antibacterial drug, trimethoprim, where the water content of the media is decreased by increasing concentrations of sorbitol. The ability of wild type and mutant clones of R67 DHFR to allow host Escherichia coli to grow in the presence of trimethoprim plus added sorbitol parallels the catalytic efficiency of the DHFR clones, indicating water content strongly correlates with the in vivo function of R67 DHFR.R67 dihydrofolate reductase, a type II DHFR, 2 catalyzes the NADPH-dependent reduction of dihydrofolate (DHF) to tetrahydrofolate. The gene for this enzyme is carried by an R-plasmid, and its presence confers resistance to the antibiotic drug, trimethoprim (TMP). Trimethoprim is a competitive inhibitor of chromosomal DHFR with a picomolar K i (1). Although R67 DHFR is not a good catalyst, it is not inhibited by TMP very well, and thus it allows cell growth in the presence of this antibacterial drug (2, 3). R67 DHFR shares no homology in sequence or structure with chromosomal DHFR and has been proposed to be a good model of a primitive enzyme (4).R67 DHFR is a homotetramer with a single active site pore; the overall structure possesses 222 symmetry as seen in Fig. 1 (5). The symmetry of the active site results in overlapping binding sites for substrate, DHF, and cofactor, NADPH. This can be observed as R67 DHFR binds a total of two ligands as follows: either two NADPH molecules or two folate/DHF molecules or one NADPH plus one folate/DHF molecule (6). The first two complexes are dead-end (binary) complexes, whereas the third is the productive ternary complex. Because of the 222 symmetry, binding to either ligand is unlikely to be optimal.The active site pore of R67 DHFR is unusual in its hourglass shape as well as its large size (2938 Å 3 for apoR67 DHFR with hydrogens added, calculated by CASTp (4, 7)). Because of this large volume, DHF and NADPH cannot occupy all the space in the pore and must use water to mediate some contacts with the protein.How do substrate and cofactor bind to R67 DHFR? From NMR, crystallography, and docking studies, the pteridine ring of DHF/fo...

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A plasmid-encoded dihydrofolate reductase from trimethoprim-resistant bacteria has a novel D2-symmetric active site

Cited by 78 publications

References 35 publications

Crystal Structure of a Type II Dihydrofolate Reductase Catalytic Ternary Complex

Crystal Structure of a Type II Dihydrofolate Reductase Catalytic Ternary Complex

Dramatic acceleration of protein folding by stabilization of a nonnative backbone conformation

A Balancing Act between Net Uptake of Water during Dihydrofolate Binding and Net Release of Water upon NADPH Binding in R67 Dihydrofolate Reductase

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