Role of the loop containing residue 115 in the induced-fit mechanism of the bacterial cell wall biosynthetic enzyme MurA

The induced-fit mechanism in Enterobacter cloacae MurA has been investigated by kinetic studies and X-ray crystallography. The antibiotic fosfomycin, an irreversible inhibitor of MurA, induced a structural change in UDP-N-acetylglucosamine (UDPGlcNAc)-liganded enzyme with a time dependence similar to that observed for the inactivation progress. The mechanism of action of fosfomycin on MurA appeared to be of the bimolecular type, the overall rate constants of inactivation and structural change being = 104 M(-1) s(-1) and = 85 M(-1) s(-1), respectively. Fosfomycin as well as the second MurA substrate, phosphoenolpyruvate (PEP), are known to interact with the side chain of Cys115. Like wild-type MurA, the catalytically inactive single-site mutant protein Cys115Ser structurally interacted with UDPGlcNAc in a rapidly reversible reaction. However, in contrast to wild-type enzyme, binding of PEP to mutant protein induced a rate-limited, biphasic structural change. Fosfomycin did not affect the structure of the mutant protein. The crystal structure of unliganded Cys115Ser MurA at 1.9 A resolution revealed that the overall conformation of the loop comprising residues 112-121 is not influenced by the mutation. However, other than Cys115 in wild-type MurA, Ser115 exhibits two distinct side-chain conformations. A detailed view on the loop revealed the existence of an elaborate hydrogen-bonding network mainly supplied by water molecules, presumably stabilizing its conformation in the unliganded state. The comparison between the known crystal structures of MurA, together with the kinetic data obtained, suggest intermediate conformational states in the MurA reaction, in which the loop undergoes multiple structural changes upon ligand binding.     

A novel inhibitor that suspends the induced fit mechanism of UDP-N-acetylglucosamine enolpyruvyl transferase (MurA)

MurA (UDP-N-acetylglucosamine enolpyruvyl transferase, EC 2.5.1.7) catalyzes the first committed step in the synthesis of the bacterial cell wall. It is the target of the naturally occurring, broad-spectrum antibiotic fosfomycin. Fosfomycin, an epoxide, is a relatively poor drug because an ever-increasing number of bacteria have developed resistance to fosfomycin. Thus, there is a critical need for the development of novel drugs that target MurA by a different molecular mode of action. We have identified a new scaffold of potent MurA inhibitors, derivatives of 5-sulfonoxy-anthranilic acid, using high-throughput screening. T6361 and T6362 are competitive inhibitors of MurA with respect to the first substrate, UDP-N-acetylglucosamine (UNAG), with a K(i) of 16 microM. The crystal structure of the MurA.T6361 complex at 2.6 angstrom resolution, together with fluorescence data, revealed that the inhibitor targets a loop, Pro112 to Pro121, that is crucial for the structural changes of the enzyme during catalysis. Thus, this new class of MurA inhibitors is not active site-directed but instead obstructs the transition from the open (unliganded) to the closed (UNAG-liganded) enzyme form. The results provide evidence for the existence of a MurA.UNAG collision complex that may be specifically targeted by small molecules different from ground-state analogs of the enzymatic reaction.     

Functional consequence of covalent reaction of phosphoenolpyruvate with UDP-N-acetylglucosamine 1-carboxyvinyltransferase (MurA)

The enzyme MurA has been an established antibiotic target since the discovery of fosfomycin, which specifically inhibits MurA by covalent modification of the active site residue Cys-115. Early biochemical studies established that Cys-115 also covalently reacts with substrate phosphoenolpyruvate (PEP) to yield a phospholactoyl adduct, but the structural and functional consequences of this reaction remained obscure. We captured and depicted the Cys-115-PEP adduct of Enterobacter cloacae MurA in various reaction states by X-ray crystallography. The data suggest that cellular MurA predominantly exists in a tightly locked complex with UDP-N-acetylmuramic acid (UNAM), the product of the MurB reaction, with PEP covalently attached to Cys-115. The uniqueness and rigidity of this "dormant" complex was previously not recognized and presumably accounts for the failure of drug discovery efforts toward the identification of novel and effective MurA inhibitors. We demonstrate that recently published crystal structures of MurA from various organisms determined by different laboratories were indeed misinterpreted and actually contain UNAM and covalently bound PEP. The Cys-115-PEP adduct was also captured in vitro during the reaction of free MurA and substrate UDP-N-acetylglucosamine or isomer UDP-N-acetylgalactosamine. The now available series of crystal structures allows a comprehensive view of the reaction cycle of MurA. It appears that the covalent reaction of MurA with PEP fulfills dual functions by tightening the complex with UNAM for the efficient feedback regulation of murein biosynthesis and by priming the PEP molecule for instantaneous reaction with substrate UDP-N-acetylglucosamine.     

Structures of Escherichia coli branched-chain amino acid aminotransferase and its complexes with 4-methylvalerate and 2-methylleucine: induced fit and substrate recognition of the enzyme.

The following three-dimensional structures of three forms of Escherichia coli branched-chain amino acid aminotransferase (eBCAT) have been determined by the X-ray diffraction method: the unliganded pyridoxal 5'-phosphate (PLP) form at a 2.1 A resolution, and the two complexes with the substrate analogues, 4-methylvalerate (4-MeVA) as the Michaelis complex model and 2-methylleucine (2-MeLeu) as the external aldimine model at 2.4 A resolution. The enzyme is a trimer of dimers, and each subunit consists of small and large domains, and the interdomain loop. The active site is formed by the residues at the domain interface and those from two loops of the other subunit of the dimer unit, and binds one PLP with its re-face directed toward the protein side. Upon binding of a substrate, Arg40 changes its side-chain direction to interact with the interdomain loop, and the loop, which is disordered in the unliganded form, shows its ordered structure on the active-site cavity, interacts with the hydrophobic side chain of the substrate, and shields it from the solvent region. The substrate binds to the active-site pocket with its alpha-hydrogen toward the protein side, its side-chain on the side of O3 of PLP, and its alpha-carboxylate on the side of the phosphate group of PLP. The hydrophobic side-chain of the substrate is recognized by Phe36, Trp126, Tyr129, Tyr164, Tyr31*, and Val109*. The alpha-carboxylate of the substrate binds to the unique site constructed by three polar groups (two main-chain NH groups of the beta-turn at Thr257 and Ala258 and the hydroxy group of Tyr95) which are activated by the access of Arg40 to the main-chain C=O group of the beta-turn and the coordination of Arg97 to the hydroxy group. Since Arg40 is the only residue that significantly changes its side-chain conformation and directly interacts with the interdomain loop and the beta-turn, the residue plays important roles in the induced fit of the interdomain loop and the alpha-carboxylate recognition of the substrate.     

Restricted backbone conformational and motional flexibilities of loops containing peptidyl-proline bonds dominate the enzyme activity of staphylococcal nuclease

The role of cis-trans isomerizations of peptidyl-proline bonds in the enzyme activity of staphylococcal nuclease (SNase) was examined by mutation of proline residues. The proline-free SNase ([Pro-]SNase), namely, P11A/P31A/P42A/P47T/P56A/P117G-mutant SNase, was adopted for elucidating the correlation between the nuclease activity and the backbone conformational and dynamic states of SNase. The 3D solution structure of [Pro-]SNase has been determined by heteronuclear NMR experiments. Comparing the structure of [Pro-]SNase with the structure of SNase revealed the conformational differences between the two proteins. In the structure of [Pro-]SNase, conformational rearrangements were observed for the loop of residues Ala112-His121 containing a trans Lys116-Gly117 peptide bond and for the C-terminal alpha-helical loop of residues Leu137-Glu142. Mutation of proline at position 117 also caused the conformational rearrangement of the p-loop (Asp77-Leu89), which is remote from the Ala112-His121 loop. The Ala112-His121 loop and p-loop are placed closer to each other in [Pro-]SNase than in SNase. The backbone dynamic features of the omega-loop (Pro42-Pro56) of SNase are different from those of [Pro-]SNase. The backbone of the omega-loop exhibits restricted flexibility with slow conformational exchange motions in SNase, but is highly flexible in [Pro-]SNase. The analysis indicates that the restrained backbone conformation of the Ala112-His121 loop and restricted flexibility of the omega-loop are two dominant factors determining the enzyme activity of SNase. Of the two factors, the former is correlated with the strained cis Lys116-Pro117 peptide bond and the latter is correlated with the cis-trans isomerizations of the His46-Pro47 peptide bond.     

Role of proline residues in the folding of serine hydroxymethyltransferase

Previous studies on the folding mechanism of Escherichia coli serine hydroxymethyltransferase (SHMT) showed that the final rate determining folding step was from an intermediate that contained two fully folded domains with N-terminal segments of approximately 55 residues and interdomain segments of approximately 50 residues that were still solvent exposed and subject to proteolysis. The interdomain segment contains 3 Pro residues near its N terminus and 2 Pro residues near its C terminus. The 5 Pro residues were each mutated to both a Gly and Ala residue, and each mutant SHMT was purified and characterized with respect to kinetic properties, stability, secondary structure, and folding mechanism. The results showed that Pro214 and Pro218 near the N terminus of the interdomain segment are not critical for folding, stability, or activity. The P216A mutant also retained most of the characteristics of the native enzyme, but its folding rate was altered. However, the P216G mutant was severely compromised in folding into a catalytically competent enzyme. Mutation of both Pro258 and Pro264 had altered folding kinetics and resulted in enzymes that expressed little catalytic activity. The Phe257-Pro258 bond is cis in its configuration, and the P258A mutant SHMT showed reduced thermal stability. Pro216, Pro258, and Pro264 are conserved in all 53 known sequences of this enzyme. The results are discussed in terms of the role of each Pro residue in maintaining the structure and function of SHMT and a possible role in pyridoxal 5'-phosphate addition to the apo-enzyme.     

Kinetic characterization of native and cysteine 112-modified glutathione S-transferase A1-1: reassessment of nonsubstrate ligand binding

We have prepared human glutathione S-transferase isoform A1-1 (GST A1-1) which has been chemically modified at cysteine 112. These modifications include formation of mixed disulfides with glutathione ("glutathiolation") and cross-linkage of the GST dimer with bis-maleimides reacting with the equivalent Cys 112 residues of the two monomers. This residue (Cys 112) lies adjacent to the hydrophobic substrate binding site, and its side chain thiol projects into the large, solvent-filled cleft which is widely reported in the literature to be the binding site of nonsubstrate ligands. Both types of modification block this intersubunit cleft region and significantly change its chemical environment. Kinetic experiments with these altered enzymes revealed that neither type of modification affects the catalytic activity of GST A1-1 or the binding of nonsubstrate ligands. The lack of an effect on glutathione conjugation activity is somewhat surprising given the proximity of cysteine 112 to the hydrophobic substrate binding site. More surprising, however, is the observation that modification at cysteine 112 has no effect on the binding of nonsubstrate ligands. Furthermore, two of these ligands, lithocholic acid and estradiol disulfate, unexpectedly exhibited competitive inhibition of the unmodified enzyme, suggesting that they bind in the hydrophobic substrate site rather than some accessory ligand binding site. Together, these results strongly argue against the intersubunit cleft as the nonsubstrate ligand binding site and prompt a reassessment of how these ligands interact with GST A1-1.     

beta-Alanyl-beta-alanine in cyclic beta-turned peptides.

In the present paper we describe the synthesis, purification, and single crystal x-ray analysis of the cyclic pentapeptide cyclo-(L-Pro-L-Pro-L-Phe-beta-Ala-beta-Ala). The peptide was synthesized by classical solution methods and the cyclization of the free pentapeptide was accomplished in good yields in diluted methylene-chloride solution using N,N-dicyclohexylcarbodiimide. The compound crystallizes in the monoclinic space group P21 from hot water with five solvent molecules. The Pro1-Pro2 peptide bond is cis and the molecular conformation is stabilized by an intramolecular hydrogen bond between the CO group of the beta-Ala5 and the NH of the Phe3 residue. The Pro1-Pro2 segment occupies the relative positions 2 and 3 of a type VIa beta-turn, while the L-phenylalanyl-beta-alanyl-beta-alanine segment is incorporated in a C13-like ring structure. The crystal packing is characterized by a network of 11 intermolecular hydrogen bonds involving all the remaining CO, NH, and the water molecules.     

Asparagine 23 and aspartate 305 are essential residues in the active site of UDP-N-acetylglucosamine enolpyruvyl transferase from Enterobacter cloacae

UDP-N-acetylglucosamine enolpyruvyl transferase (MurA) catalyzes the transfer of the intact enolpyruvyl moiety of phosphoenolpyruvate (PEP) to the 3'-hydroxyl group of UDP-N-acetylglucosamine (UDPNAG). This reaction constitutes the first committed step in the biosynthesis of the bacterial cell wall component peptidoglycan (murein). The transfer reaction involves the nucleophilic attack of the 3'-hydroxyl group of UDPNAG at the C-2 of PEP. The three-dimensional structure of MurA complexed with UDPNAG revealed an aspartate residue (D305 in the En. cloacae sequence) close to the 3'-hydroxyl group of UDPNAG, suggesting that it may act as an acid-base catalyst in the active center of the enzyme. In addition to aspartate 305, asparagine 23 also interacts with the 3'-hydroxyl group; however, its role in catalysis or binding of the UDPNAG substrate is unclear. To gain information on the role of these two amino acids in the MurA-catalyzed reaction we have exchanged D305 for alanine, cysteine, histidine, and glutamate, and N23 for alanine and serine using site-directed mutagenesis. While the D305 alanine, cysteine, and histidine mutant proteins do not have detectable enzymatic activity, the D305E mutant protein exhibits a low residual activity (ca. 0.1% of the wild-type enzyme). Unlike with wild-type MurA, no exothermic signal was obtained when the D305A and -E mutant proteins were titrated with UDPNAG, demonstrating that the affinity of the sugar nucleotide substrate is reduced as a result of the amino acid exchange. The reduced affinity to UDPNAG leads to a lower propensity of C115 to form either the O-phosphothioketal with PEP or the thioether with the antibiotic fosfomycin. These findings emphasize the dual role of D305 as a general base and an essential binding partner to UDPNAG in the active site of MurA. Similarly, the two N23 mutant proteins showed a much lower catalytic activity although binding of UDPNAG was not as much affected as in the case of the D305 mutant proteins. This result indicates that this amino acid residue is mainly involved in stabilization of transition states.     

Crystal structures of a phosphotransacetylase from <Emphasis Type="Italic">Bacillus subtilis</Emphasis> and its complex with acetyl phosphate

Phosphotransacetylase (Pta) [EC 2.3.1.8] plays a major role in acetate metabolism by catalyzing the reversible transfer of the acetyl group between coenzyme A (CoA) and orthophosphate: CH(3)COSCoA+HPO(4)(2-)<-->CH(3)COOPO(3)(2-) +CoASH. In this study, we report the crystal structures of Pta from Bacillus subtilis at 2.75 A resolution and its complex with acetyl phosphate, one of its substrates, at 2.85 A resolution. In addition, the Pta activity of the enzyme has been assayed. The enzyme folds into an alpha/beta architecture with two domains separated by a prominent cleft, very similar to two other known Pta structures. The enzyme-acetyl phosphate complex structure reveals a few potential substrate binding sites. Two of them are located in the middle of the interdomain cleft: each one is surrounded by a region of strictly and highly conserved residues. High structural similarities are found with 4-hydroxythreonine-4-phosphate dehydrogenase (PdxA), and isocitrate and isopropylmalate dehydrogenases, all of which utilize NADP+ as their cofactor, which binds in the interdomain cleft. Their substrate binding sites are close to the acetyl phosphate binding sites of Pta in the cleft as well. These results suggest that the CoA is likely to bind to the interdomain cleft of Pta in a similar way as NADP+ binds to the other three enzymes.     

Peptide substrates for chymosin (rennin). Interaction sites in kappa-casein-related sequences located outside the (103-108)-hexapeptide region that fits into the enzyme''s active-site cleft.

The role of individual amino acid residues in the 98-102 and 111-112 regions of bovine kappa-casein in its interaction with the milk-clotting enzyme chymosin (rennin) was investigated. to this end the tryptic 98-112 fragment of kappa-casein was modified in its N- and/or C-terminal part by chemical (guanidation, ethoxyformylation, repeated Edman degradation) and enzymic (carboxypeptidase) treatments. Further, use was made of short synthetic kappa-casein analogues in which His-102 had been replaced by Pro or Lys. All peptides and their derivatives were tested comparatively at various pH values for their ability to act as chymosin substrates via specific cleavage of the peptide bond at position 105-106. The results indicate that in the alternating 98-102 sequence (His-Pro-His-Pro-His) the His as well as the Pro residues contribute to the substrate activity with no predominant role of any one of these groups. Another interaction site is formed by the Lys residue at position 111 of the substrate. A model of the enzyme-substrate complex is proposed. Herein the 103-108 fragment of the substrate, to be accommodated within the enzyme's active-site cleft, is brought into position by electrostatic binding (via His-98, His-100, His-102 and Lys-111) near the entrance of the cleft. These interactions are strongly supported by Pro residues at positions 99, 101, 109 and 110 of the substrate, which act as stabilizers of the proper conformation of the substrate in the enzyme-substrate complex.     

Structure of the UDP-glucosyltransferase GtfB that modifies the heptapeptide aglycone in the biosynthesis of vancomycin group antibiotics

BACKGROUND: Members of the vancomycin group of glycopeptide antibiotics have an oxidatively crosslinked heptapeptide scaffold decorated at the hydroxyl groups of 4-OH-Phegly4 or beta-OH-Tyr6 with mono- (residue 6) or disaccharides (residue 4). The disaccharide in vancomycin itself is L-vancosamine-1,2-glucose, and in chloroeremomycin it is L-4-epi-vancosamine-1,2-glucose. The sugars and their substituents play an important role in efficacy, particularly against vancomycin-resistant pathogenic enterococci. RESULTS: The glucosyltransferase, GtfB, that transfers the glucose residue from UDP-glucose to the 4-OH-Phegly4 residue of the vancomycin aglycone, initiating the glycosylation pathway in chloroeremomycin maturation, has been crystallized, and its structure has been determined by X-ray analysis at 1.8 A resolution. The enzyme has a two-domain structure, with a deep interdomain cleft identified as the likely site of UDP-glucose binding. A hydrophobic patch on the surface of the N-terminal domain is proposed to be the binding site of the aglycone substrate. Mutagenesis has revealed Asp332 as the best candidate for the general base in the glucosyltransfer reaction. CONCLUSIONS: The structure of GtfB places it in a growing group of glycosyltransferases, including Escherichia coli MurG and a beta-glucosyltransferase from T4 phage, which together form a subclass of the glycosyltransferase superfamily and give insights into the recognition of the NDP-sugar and aglycone cosubstrates. A single major interdomain linker between the N- and C- terminal domains suggests that reprogramming of sugar transfer or aglycone recognition in the antibiotic glycosyltransferases, including the glycopeptide and also the macrolide antibiotics, will be facilitated by this structural information.     

Alternative structural state of transferrin. The crystallographic analysis of iron-loaded but domain-opened ovotransferrin N-lobe

Transferrins bind Fe3+ very tightly in a closed interdomain cleft by the coordination of four protein ligands (Asp60, Tyr92, Tyr191, and His250 in ovotransferrin N-lobe) and of a synergistic anion, physiologically bidentate CO32-. Upon Fe3+ uptake, transferrins undergo a large scale conformational transition: the apo structure with an opening of the interdomain cleft is transformed into the closed holo structure, implying initial Fe3+ binding in the open form. To solve the Fe3+-loaded, domain-opened structure, an ovotransferrin N-lobe crystal that had been grown as the apo form was soaked with Fe3+-nitrilotriacetate, and its structure was solved at 2.1 A resolution. The Fe3+-soaked form showed almost exactly the same overall open structure as the iron-free apo form. The electron density map unequivocally proved the presence of an iron atom with the coordination by the two protein ligands of Tyr92-OH and Tyr191-OH. Other Fe3+ coordination sites are occupied by a nitrilotriacetate anion, which is stabilized through the hydrogen bonds with the peptide NH groups of Ser122, Ala123, and Gly124 and a side chain group of Thr117. There is, however, no clear interaction between the nitrilotriacetate anion and the synergistic anion binding site, Arg121.     

Evidence that the fosfomycin target Cys115 in UDP-N-acetylglucosamine enolpyruvyl transferase (MurA) is essential for product release

MurA (UDP-N-acetylglucosamine enolpyruvyl transferase, EC 2.5.1.7) is an essential enzyme in the biosynthesis of the peptidoglycan layer of the bacterial cell. It provides an attractive template for the design of novel antibiotic drugs and is the target of the naturally occurring antibiotic fosfomycin, which covalently attaches to Cys115 in the active site of the enzyme. Mutations of Cys115 to Asp exist in pathogens such as Mycobacteria or Chlamydia rendering these organisms resistant to fosfomycin. Thus, there is a need for the elucidation of the role of this cysteine in the MurA reaction. We determined the x-ray structure of the C115S mutant of Enterobacter cloacae MurA, which was crystallized in the presence of the substrates of MurA. The structure depicts the product state of the enzyme with enolpyruvyl-UDP-N-acetylglucosamine and inorganic phosphate trapped in the active site. Kinetic analysis revealed that the Cys-to-Ser mutation results in an enzyme that appears to perform a single turnover of the reaction. Opposing the common view of Cys115 as a key residue in the chemical reaction of enolpyruvyl transfer, we now conclude that the wild-type cysteine is essential for product release only. On the basis of a detailed comparison of the product state with the intermediate state and an unliganded state of MurA, we propose that dissociation of the products is an ordered event with inorganic phosphate leaving first. Phosphate departure appears to trigger a suite of conformational changes, which finally leads to opening of the two-domain structure of MurA and the release of the second product enolpyruvyl-UDP-N-acetylglucosamine.     

Crystal structures of mouse class II alcohol dehydrogenase reveal determinants of substrate specificity and catalytic efficiency

The structure of mouse class II alcohol dehydrogenase (ADH2) has been determined in a binary complex with the coenzyme NADH and in a ternary complex with both NADH and the inhibitor N-cyclohexylformamide to 2.2 A and 2.1 A resolution, respectively. The ADH2 dimer is asymmetric in the crystal with different orientations of the catalytic domains relative to the coenzyme-binding domains in the two subunits, resulting in a slightly different closure of the active-site cleft. Both conformations are about half way between the open apo structure and the closed holo structure of horse ADH1, thus resembling that of ADH3. The semi-open conformation and structural differences around the active-site cleft contribute to a substantially different substrate-binding pocket architecture as compared to other classes of alcohol dehydrogenase, and provide the structural basis for recognition and selectivity of alcohols and quinones. The active-site cleft is more voluminous than that of ADH1 but not as open and funnel-shaped as that of ADH3. The loop with residues 296-301 from the coenzyme-binding domain is short, thus opening up the pocket towards the coenzyme. On the opposite side, the loop with residues 114-121 stretches out over the inter-domain cleft. A cavity is formed below this loop and adds an appendix to the substrate-binding pocket. Asp301 is positioned at the entrance of the pocket and may control the binding of omega-hydroxy fatty acids, which act as inhibitors rather than substrates. Mouse ADH2 is known as an inefficient ADH with a slow hydrogen-transfer step. By replacing Pro47 with His, the alcohol dehydrogenase activity is restored. Here, the structure of this P47H mutant was determined in complex with NADH to 2.5 A resolution. His47 is suitably positioned to act as a catalytic base in the deprotonation of the substrate. Moreover, in the more closed subunit, the coenzyme is allowed a position closer to the catalytic zinc. This is consistent with hydrogen transfer from an alcoholate intermediate where the Pro/His replacement focuses on the function of the enzyme.     

Crystal structure of the sulfotransferase domain of human heparan sulfate N-deacetylase/ N-sulfotransferase 1

Heparan sulfate N-deacetylase/N-sulfotransferase (HSNST) catalyzes the first and obligatory step in the biosynthesis of heparan sulfates and heparin. The crystal structure of the sulfotransferase domain (NST1) of human HSNST-1 has been determined at 2.3-A resolution in a binary complex with 3'-phosphoadenosine 5'-phosphate (PAP). NST1 is approximately spherical with an open cleft, and consists of a single alpha/beta fold with a central five-stranded parallel beta-sheet and a three-stranded anti-parallel beta-sheet bearing an interstrand disulfide bond. The structural regions alpha1, alpha6, beta1, beta7, 5'-phosphosulfate binding loop (between beta1 and alpha1), and a random coil (between beta8 and alpha13) constitute the PAP binding site of NST1. The alpha6 and random coil (between beta2 and alpha2), which form an open cleft near the 5'-phosphate of the PAP molecule, may provide interactions for substrate binding. The conserved residue Lys-614 is in position to form a hydrogen bond with the bridge oxygen of the 5'-phosphate.     

Unusual conformational changes in 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase as revealed by X-ray crystallography and NMR

The crystal structure of Escherichia coli 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK) in complex with MgADP has been determined at 1.5-A resolution with a crystallographic R factor of 0.191. The solution structure of HPPK in complex with Mg(2+) and beta,gamma-methyleneadenosine 5'-triphosphate (MgAMPPCP) has been determined using a simulated annealing protocol with 3,523 experimental NMR restraints. The root mean square deviation of the ensemble of 20 refined conformers that represent the solution structure from the mean coordinate set derived from them is 0.74 +/- 0.26 A for all backbone atoms and 0.49 +/- 0.22 A when residues Pro(14), Pro(44)-Gln(50), and Arg(84)-Pro(91) are excluded. Binding of MgADP causes significant changes in the conformation and dynamical property of three loops of HPPK that are involved in catalysis. A dramatic, unusual conformational change is that loop 3 moves away from the active center significantly with some residues moving by >17 A. The binding of MgADP also stabilizes loop 1 and loop 3 but makes loop 2 more mobile. Very similar conformational and dynamical changes are observed in the NMR solution structure of HPPK.MgAMPPCP. The conformational and dynamical changes may play important roles in both substrate binding and product release in the catalytic cycle.     

The 1.49 A resolution crystal structure of PsbQ from photosystem II of Spinacia oleracea reveals a PPII structure in the N-terminal region

We report the high-resolution structure of the spinach PsbQ protein, one of the main extrinsic proteins of higher plant photosystem II (PSII). The crystal structure shows that there are two well-defined regions in PsbQ, the C-terminal region (residues 46-149) folded as a four helix up-down bundle and the N-terminal region (residues 1-45) that is loosely packed. This structure provides, for the first time, insights into the crucial N-terminal region. First, two parallel beta-strands cross spatially, joining the beginning and the end of the N-terminal region of PsbQ. Secondly, the residues Pro9-Pro10-Pro11-Pro12 form a left-handed helix (or a polyproline type II (PPII) structure), which is stabilized by hydrogen bonds between the Pro peptide carbonyl groups and solvent water molecules. Thirdly, residues 14-33 are not visible in the electron density map, suggesting that this loop might be very flexible and presumably extended when PsbQ is free in solution. On the basis of the essential role of the N-terminal region of PsbQ in binding to PSII, we propose that both the PPII structure and the missing loop are key secondary structure elements in the recognition of specific protein-protein interactions between PsbQ and other oxygen-evolving complex extrinsic and/or intrinsic proteins of PSII. In addition, the PsbQ crystal coordinates two zinc ions, one of them is proposed to have a physiological role in higher plants, on the basis of the full conservation of the ligand protein residues in the sequence subfamily.     

Inhibitory mechanism of the Qβ lysis protein A2

The lysis protein A₂, present as a single copy on the surface of Qβ virion particles, was previously shown to inhibit the activity of MurA, an enzyme that catalyses the first committed step of murein biosynthesis. Here we report experiments with a two‐hybrid study that indicates A₂ and MurA interact directly. Moreover, experiments with a soluble MBP–A₂ fusion indicate that the interaction between MurA and A₂ is dependent on a substrate‐induced conformational change featured in the UDP‐NAG‐liganded state of MurA but not the tetrahedral intermediate state. Moreover, based on the location of L138Q, the original dominant A₂‐resistant mutant that identified MurA as the target, a directed mutagenesis strategy has identified a continuous surface required for A₂ binding. This surface spans the catalytic loop/cleft and encompasses both the catalytic and C‐terminal domains. These data support a model in which A₂ preferentially binds MurA liganded with UDP‐NAG, thereby preventing catalysis by occluding PEP from accessing the active site.     

Solvent interaction of a Hsp70 chaperone substrate-binding domain investigated with water-NOE NMR experiments

The interaction of solvent of the substrate binding domain of the bacterial heat shock 70 chaperone protein DnaK was studied in its apo form and with bound hydrophobic substrate peptide, using refined nuclear magnetic resonance experiments. Distinct differences between the two states of the protein were observed. According to our data, the apo form interacts more extensively with solvent than the peptide-bound form. Significantly, the open hydrophobic substrate binding cleft of DnaK in the apo form is found to contain several molecules of water which are displaced by the binding of the hydrophobic substrate, the peptide NRLLLTG. The solvent in the hydrophobic cleft has a residence time longer than 400 ps. It is predicted that the displacement of this trapped water must contribute to the binding free energy of the natural hydrophobic substrates of this class of protein-folding chaperone proteins.