Main-chain Nitrogen Research Articles

Structural determinants of protein dynamics: analysis of 15N NMR relaxation measurements for main-chain and side-chain nuclei of hen egg white lysozyme.

15N-labeled hen lysozyme has been studied by 2D and 3D NMR in order to characterize its dynamic behavior. The resonances of all main-chain amide nitrogen atoms were assigned, as were resonances of nitrogen atoms in 28 side chains. Relaxation measurements for the main-chain and arginine and tryptophan side-chain 15N nuclei used standard methods, and those for the 15N nuclei of asparagine and glutamine side chains used pulse sequences designed to remove unwanted relaxation pathways in the NH2 groups. The calculated order parameters (S2) show that the majority of main-chain amides undergo only small amplitude librational motions on a fast time scale (S2 > or = 0.8). Increased main-chain motion (0.5 < S2 < 0.8) is observed for a total of 19 residues located at the C-terminus, in loop and turn regions, and in the first strand of the main beta-sheet. Order parameters derived for the side chains range from 0.05 to 0.9; five of the six tryptophan residues have high order parameters (S2 > or = 0.8), consistent with their location in the closely packed core of the protein, whereas the order parameters between 0.05 and 0.3 for arginine residues confirm increased side-chain mobility at the protein surface. Order parameters for the side chains of asparagine and glutamine residues range from 0.2 to 0.8; high values are found for side chains that have low solvent accessible surfaces and well-defined chi 1 values, as measured by 3J alpha beta coupling constants. Many of the main-chain and side-chain groups with low order parameters have higher than average temperature factors in X-ray crystal structures and increased positional uncertainty in NMR solution structures. They also tend to lack persistent hydrogen bond interactions and protection against amide hydrogen exchange. The most significant correlations are found between residues with low order parameters and high surface accessibility in both crystal and solution structures. The results suggest that a lack of van der Waals contacts is a major determinant of side-chain and main-chain mobility in proteins.

Crystal structure and mutational analysis of human uracil-DNA glycosylase: Structural basis for specificity and catalysis

Crystal structures of the DNA repair enzyme human uracil-DNA glycosylase (UDG), combined with mutational analysis, reveal the structural basis for the specificity of the enzyme. Within the classic α/β fold of UDG, sequence-conserved residues form a positively charged, active-site groove the width of duplex DNA, at the C-terminal edge of the central four-stranded parallel β sheet. In the UDG-6-aminouracil complex, uracil binds at the base of the groove within a rigid preformed pocket that confers selectivity for uracil over other bases by shape complementarity and by main chain and Asn-204 side chain hydrogen bonds. Main chain nitrogen atoms are positioned to stabilize the oxyanion intermediate generated by His-268 acting via nucleophilic attack or general base mechanisms. Specific binding of uracil flipped out from a DNA duplex provides a structural mechanism for damaged base recognition.

A metastable state of Torpedo californica acetylcholinesterase generated by modification with organomercurials.

Chemical modification of Torpedo californica acetylcholinesterase by various sulfhydryl reagents results in its conversion to one of two principal states. One of these states, viz., that produced by disulfides and alkylating agents, is stable. The second state, produced by mercury derivatives, is metastable. At room temperature, it converts spontaneously, with a half-life of ca. 1 h, to a stable state similar to that produced by the disulfides and alkylating agents. Demodification of acetylcholinesterase freshly modified by mercurials, by its exposure to reduced glutathione, causes rapid release of the bound mercurial, with concomitant recovery of most of the enzymic activity of the native enzyme. In contrast, similar demodification of acetylcholinesterase modified by disulfides yields no detectable recovery of enzymic activity. Spectroscopic measurements, employing CD, intrinsic fluorescence, and binding of 1-anilino-8-naphthalenesulfonate, show that the state produced initially by mercurials is "native-like", whereas that produced by disulfides and alkylating agents, and after prolonged incubation of the mercurial-modified enzyme, is partially unfolded and displays many of the features of the "molten globule" state. Arrhenius plots show that the quasi-native state produced by organomercurials is separated by a low (5 kcal/mol) energy barrier from the native state, whereas the partially unfolded state is separated from the quasi-native state by a high energy barrier (ca. 50 kcal/mol). Comparison of the 3D structures of native acetylcholinesterase and of a heavy-atom derivative obtained with HgAc2 suggests that the mercurial-modified enzyme may be stabilized by additional interactions of the mercury atom attached to the free thiol group of Cys231, specifically with Ser228O gamma with the main-chain nitrogen and carbonyl oxygen of the same serine residue.

Satisfying Hydrogen Bonding Potential in Proteins

We have analysed the frequency with which potential hydrogen bond donors and acceptors are satisfied in protein molecules. There are a small percentage of nitrogen or oxygen atoms that do not form hydrogen bonds with either solvent or protein atoms, when standard criteria are used. For high resolution structures 9·5% and 5·1% of buried main-chain nitrogen and oxygen atoms, respectively, fail to hydrogen bond under our standard criteria, representing 5·8% and 2·1% of all main-chain nitrogen and oxygen atoms. We find that as the resolution of the data improves, the percentages fall. If the hydrogen bond criteria are relaxed many of these unsatisfied atoms form weak hydrogen bonds. However, there remain some buried atoms (1·3% NH and 1·8% CO) that fail to hydrogen bond without any immediately obvious compensating interactions.

Structure of a Phosphonate-inhibited β-Lactamase: An Analog of the Tetrahedral Transition State/Intermediate of β-Lactam Hydrolysis

The crystal structure of β-lactamase from Staphylococcus aureus inactivated by p -nitrophenyl[[ N-(benzyloxycarbonyl)amino]methyl]phosphonate, a methylphosphonate monoester monoanion inhibitor, has been determined and refined at 2·3 Å resolution. The structure reveals a tetrahedral phosphorus covalently bonded to the O γ atom of the active site serine, Ser70. One of the oxygen atoms bonded to phosphorus is located in the oxyanion hole formed by the two main-chain nitrogen atoms of Ser70 and Gln237, and the second bonded oxygen is solvated. The (benzyloxycarbonyl)aminomethyl group is oriented towards the active site gully such that the peptide group forms compensating electrostatic interactions with polar groups on the enzyme. The benzyl group forms a hydrophobic interaction with Ile239 and an aromatic-aromatic edge-to-face interaction with Tyr105, which has undergone a conformational transition relative to the native structure. The mode of binding supports the proposal that on reaction with the enzyme, the phosphonate generates a structure analogous to the tetrahedral transition state/intermediate associated with the acylation step of a normal substrate. The disposition of the phosphonyl group in this complex is the same as that of the corresponding phosphoryl group in the complex resulting from the inhibition of trypsin by diisopropylphosphofluoridate. The structure is consistent with a mechanism of inactivation that follows an associative pathway, proceeding via a transition state/intermediate in which phosphorus is penta-co-ordinated, forming a trigonal bipyramidal geometry with the phosphonyl donor ( p-nitrophenol) and acceptor (Ser70 O γ atom) in apical positions. A model of this transition state can be accommodated in the active site of β-lactamase without any steric hindrance. A model of the tetrahedral transition state associated with the acylation step by benzyl penicillin has been derived. Because of the conformational rigidity of the fused rings of penicillin molecules, the orientation of the substrate is fixed once the tetrahedral carbonyl carbon and its ligands are superimposed on the phosphonate group. The outcome is that the carboxylate substituent on the thiazolidine ring forms a salt bridge with Lys234, and the preferred puckering of the ring is that observed in the crystal structure of ampicillin, the so-called "open" conformer.

How to perform small peptide cyclizations

Small cyclopeptides of four to six residues are very interesting for their biological properties. Unfortunately, the synthesis of the linear precursor is generally fastidious and the cyclization often occurs in low yields. Molecular modeling used through the genmol program is a powerful tool for predicting the best precursor, as was shown in a previous paper about five tetrapeptides. However, sometimes all the linear precursors of a cyclopeptide can be unfavorable for cyclization when no structural feature ( N-Me amino acid. Pro, D-amino acid) is present in the peptide. This led us to develop a method using a reversible chemical modification of the peptide main chain in order to favor the cisoid conformation able to cyclize easily. Tetraphenylalanine was used as a model, with the tert-butyloxycarbonyl (Boc) group as substituent on the main-chain nitrogen atoms. The cyclization yield increases from less than 1 % to 27% after this chemical modification and cleavage of the Boc groups. Molecular modeling on such molecules shows that this yield increase is due to a preferred conformation having the terminal functions close together induced by the Boc substituents.

Structure of the complex of proteinase K with a substrate analogue hexapeptide inhibitor at 2.2-A resolution

The crystal structure of a transition state/product complex formed by the interaction between proteinase K and the substrate analogue N-Ac-L-Pro-L-Ala-L-Pro-L-Phe-D-Ala-L-Ala-NH2 has been determined at a resolution of 2.2 A and refined to an R-factor of 0.165 for 12,725 reflections. The inhibitor forms a stable complex through a series of hydrogen bonds with protein atoms and water molecules. The inhibitor is hydrolyzed between Phe 4I and D-Ala5I (I indicates inhibitor). The two fragments are separated by a distance of 3.07 A between the carbonyl carbon and the main chain nitrogen. Both fragments remain bound to the protein. The N-terminal fragment occupies subsites S5 to S1, whereas the C-terminal part is bound in S1' and S2', the first time that electron density for a substrate analogue has been observed in the P1' and P2' sites of a subtilisin-like enzyme. The flexible segments of the substrate recognition sites Gly100-Tyr104 and Ser132-Gly136 move appreciably to accommodate the inhibitor. Biochemical results indicate an inhibition by this specifically designed peptide of 95%.

Structure of oxidized bacteriophage T4 glutaredoxin (thioredoxin): Refinement of native and mutant proteins

The structure of wild-type bacteriophage T4 glutaredoxin (earlier called thioredoxin) in its oxidized form has been refined in a monoclinic crystal form at 2.0 Å resolution to a crystallographic R-factor of 0.209. A mutant T4 glutaredoxin gives orthorhombic crystals of better quality. The structure of this mutant has been solved by molecular replacement methods and refined at 1.45 Å to an R-value of 0.175. In this mutant glutaredoxin, the active site residues Val15 and Tyr16 have been substituted by Gly and Pro, respectively, to mimic that of Escherichia coli thioredoxin. The main-chain conformation of the wild-type protein is similar in the two independently determined molecules in the asymmetric unit of the monoclinic crystals. On the other hand, side-chain conformations differ considerably between the two molecules due to heterologous packing interactions in the crystals. The structure of the mutant protein is very similar to the wild-type protein, except at mutated positions and at parts involved in crystal contacts. The active site disulfide bridge between Cys14 and Cys17 is located at the first turn of helix α1. The torsion angles of these residues are similar to those of Escherichia coli thioredoxin. The torsion angle around the SS bond is smaller than that normally observed for disulfides: 58 °, 67 ° and 67 ° for wild-type glutaredoxin molecule A and B and mutant glutaredoxin, respectively. Each sulfur atom of the disulfide cysteines in T4 glutaredoxin forms a hydrogen bond to one main-chain nitrogen atom. The active site is shielded from solvent on one side by the β-carbon atoms of the cysteine residues plus side-chains of residues 7, 9, 21 and 33. From the opposite side, there is a cleft where the sulfur atom of Cys14 is accessible and can be attacked by a nucleophilic thiolate ion in the initial step of the reduction reaction.

Inhibition of β-lactamase by clavulanate: Trapped intermediates in cryocrystallographic studies

Crystallographic studies of the complex between β-lactamase and clavulanate reveal a structure of two acylenzymes with covalent bonds at the active site Ser70, representing two different stages of inhibitor degradation alternately occupying the active site. Models that are consistent with biochemical data are derived from the electron density map and refined at 2·2 Å resolution: cis enamine, in which the carboxylate group of the clavulanate molecule makes a salt bridge with Lys234 of β-lactamase; decarboxylated trans enamine, which is oriented away from Lys234. For both acylenzymes, the carbonyl oxygen atom of the ester group occupies the oxyanion hole in a manner similar to that found in inhibitor binding to serine proteases. Whereas the oxygen atom in the trans product is optimally positioned in the oxyanion hole, that of the cis product clashes with the main-chain nitrogen atom of Ser70 and the β-carbon atom of the adjacent Ala69. In contrast to cis to trans isomerization in solution that relieves the steric strain inherent in a cis double bond, at the enzyme-inhibitor interface two additional factors play an important role. The salt bridge enhances the stability of the cis product, while the steric strain introduced by the short contacts with the protein reduces its stability.

Structure of the histidine-containing phosphocarrier protein HPr from Bacillus subtilis at 2.0-A resolution.

The crystal structure of the histidine-containing phosphocarrier protein (HPr) of the phosphoenolpyruvate:sugar phosphotransferase system (PTS) from Bacillus subtilis has been determined at 2.0-A resolution and refined to a crystallographic residual error R-factor of 0.150. The secondary-structure folding topology of the molecule is that of an open-face beta-sandwich formed by four antiparallel beta-strands packed against three alpha-helices. The active-site histidine, His-15, caps the N terminus of the first helix, suggesting that the helix dipole plays a role in stabilizing the phosphorylated state of the histidine. A sulfate anion located between His-15 and the neighboring Arg-17 has been identified in the electron-density map. Association of this negatively charged species with the two key catalytic residues implies that the crystal structure resembles the phosphorylated state of the protein. A model of the phosphorylated form of the molecule is proposed, in which the negatively charged phosphoryl group interacts with two main-chain nitrogen atoms of the following helix and with the guanidinium group of Arg-17. It is also proposed that the phosphoryl transfer from HPr to the IIA domain of the glucose permease involves Arg-17 switching between two salt bridges: one with the phosphorylated histidyl of HPr and the other with two aspartyl residues associated with the active site of the IIA domain of glucose permease, which are accessible upon complex formation.

Crystal structure of the binary complex of pig muscle phosphoglycerate kinase and its substrate 3-phospho-D-glycerate.

Pig muscle phosphoglycerate kinase has been crystallized from polyethyleneglycol in the presence of its substrate 3-phospho-D-glycerate (3-PG) and the structure has been determined at 2.0 A resolution. The structure was solved using the known structure of the substrate-free horse muscle enzyme and has been refined to a crystallographic R-factor of 21.5%. 3-Phospho-D-glycerate is bound to the N-domain of the enzyme through a network of hydrogen bonds to a cluster of basic amino acid residues and by electrostatic interactions between the negatively charged phosphate and these basic protein side chains. This binding site is in good agreement with earlier proposals [Banks et al., Nature (London) 279:773-777, 1979]. The phosphate oxygen atoms are hydrogen bonded to His-62, Arg-65, Arg-122, and Arg-170. The 2-hydroxyl group, which defines the D-isomer of 3PG, is hydrogen bonded to Asp-23 and Asn-25. The carboxyl group of 3-PG points away from the N-domain towards the C-domain and is hydrogen bonded via a water molecule to main chain nitrogen atoms of helix-14. The present structure of the 3-PG-bound pig muscle enzyme is compared with the structure of the substrate-free horse enzyme. Major changes include an ordering of helix-13 and a domain movement, which brings the N-domain closer to the ATP-binding C-domain. This domain movement consists of a 7.7 degree rotation, which is less than previously estimated for the ternary complex. Local changes close to the 3-PG binding site include an ordering of Arg-65 and a shift of helix-5.

Structure of the IIA domain of the glucose permease of Bacillus subtilis at 2.2-.ANG. resolution

The crystal structure of the IIA domain of the glucose permease of the phosphoenolpyruvate:sugar phosphotransferase system (PTS) from Bacillus subtilis has been determined at 2.2-A resolution. Refinement of the structure is in progress, and the current R-factor is 0.201 (R = sigma h parallel Fo magnitude of - Fc parallel/sigma h magnitude of Fo, where magnitude of Fo and magnitude of Fc are the observed and calculated structure factor amplitudes, respectively) for data between 6.0- and 2.2-A resolution for which F greater than or equal to 2 sigma (F). This is an antiparallel beta-barrel structure that incorporates "Greek key" and "jellyroll" topological motifs. A shallow depression is formed at the active site by part of the beta-sheet and an omega-loop flanking one side of the sheet. His83, the histidyl residue which is the phosphorylation target of HPr and which transfers the phosphoryl group to the IIB domain of the permease, is located at the C-terminus of a beta-strand. The N epsilon atom is partially solvated and also interacts with the N epsilon atom of a second histidyl residue, His68, located at the N-terminus of an adjacent beta-strand, suggesting they share a proton. The geometry of the hydrogen bond is imperfect, though. Electrostatic interactions with other polar groups and van der Waals contacts with the side chains of two flanking phenylalanine residues assure the precise orientation of the imidazole rings. The hydrophobic nature of the surface around the His83-His68 pair may be required for protein-protein recognition by HPr or/and by the IIB domain of the permease. The side chains of two aspartyl residues, Asp31 and Asp87, are oriented toward each other across a narrow groove, about 7 A from the active-site His83, suggesting they may play a role in protein-protein interaction. A model of the phosphorylated form of the molecule is proposed, in which oxygen atoms of the phosphoryl group interact with the side chain of His68 and with the main-chain nitrogen atom of a neighboring residue, Val89. The model, in conjunction with previously reported site-directed mutagenesis experiments, suggests that the phosphorylation of His83 may be accompanied by the protonation of His68. This may be important for the interaction with the IIB domain of the permease and/or play a catalytic role in the phosphoryl transfer from IIA to IIB.

Refined crystal structure of β-lactamase from Staphylococcus aureus PC1 at 2.0 Å resolution

The crystal structure of a class A β-lactamase from Staphylococcus aureus PC1 has been refined at 2.0 Å resolution. The resulting crystallographic R-factor ( R = ∑ h∥F o ¦−¦F c ∥/∑ h¦F o ¦, where ¦F o ¦ and ¦F c ¦ are the observed and calculated structure factor amplitudes, respectively), is 0.163 for the 17,547 reflections with I ≥ 2 σ( I) within the 8.0 Å to 2.0 Å resolution range. The molecule consists of two closely associated domains. One domain is formed by a five-stranded antiparallel β-sheet with three helices packing against a face of the sheet. The second domain is formed mostly by helices that pack against the second face of the sheet. The active site is located in the interface between the two domains, and many of the residues that form it are conserved in all known sequences of class A β-lactamases. Similar to the serine proteases, an oxyanion hole is implicated in catalysis. It is formed by two main-chain nitrogen atoms, that of the catalytic seryl residue, Ser70, and that of Gln237 on an edge β-strand of the major β-sheet. Ser70 is interacting with another conserved seryl residue, Ser130, located between the two ammonium groups of the functionally important lysine residues, Lys73 and Lys234. Such intricate interactions point to a possible catalytic role for this second seryl residue. Another key catalytic residue is Glu166. There are several unusual structural features associated with the active site. (1) A cis peptide bond has been identified between the catalytic Glu166 and Ile167. (2) Ala69 and Leu220 have strained φ;, ψ dihedral angles making close contacts that restrict the conformation of the active site β-strand involved in the formation of the oxyanion hole. (3) A buried aspartate residue, the conserved Asp233, is located next to the active site Lys234. It is interacting with another buried aspartyl residue, Asp246. An internal solvent molecule is also involved, but the rest of its interactions with the protein indicate it is not a cation. (4) Another conserved aspartyl residue that is desolvated is Asp131, adjacent to Ser130. Its charge is stabilized by interactions with four main-chain nitrogen atoms. (5) An internal cavity underneath the active site depression is filled with six solvent molecules. This, and an adjacent cavity occupied by three solvent molecules partially separate the Ω-loop associated with the active site from the rest of the protein. A total of 207 solvent molecules have been identified in the crystal. Of these, several are located in the active site. In particular, one water molecule occupies the oxyanion hole in a manner found in some of the serine protease structures. A second water molecule is located between Glu166, Ser70 and Asn170, indicating that it may play a role in the deacylation of a hydrolyzed substrate.

Structure of tyrosyl-tRNA synthetase refined at 2.3 Å resolution: Interaction of the enzyme with the tyrosyl adenylate intermediate

The crystal structure of tyrosyl-tRNA synthetase (EC 6.1.1.1) from Bacillus stearothermophilus has been refined to a crystallographic R-factor of 22.6% at 2.3 Å resolution using a restrained least-squares procedure. In the final model the root-mean-square deviation from ideality for bond distances is 0.018 Å and for angle distances is 0.044 Å. Each monomer consists of three domains: an α β domain (residues 1 to 220) containing a six-stranded β-sheet, an α-helical domain (248 to 318) containing five helices, and a disordered C-terminal domain (319 to 418) for which the electron density is very weak and where it has not been possible to trace the polypeptide chain. Complexes of the enzyme with the catalytic intermediate tyrosyl adenylate and the inhibitor tyrosinyl adenylate have also been refined to R-factors of 23.9% at 2.8 Å resolution and 21.0% at 2.7 Å resolution, respectively. Formation of the complexes results in some crystal cracking, but there is no significant difference in the conformation of the polypeptide chain of the three structures described here. The relative orientation of the α β and α-helical domains is similar to that previously observed for the “A” subunit of a deletion mutant lacking the C-terminal domain. Differences between these structures are confined to surface loops that are involved in crystal packing. Tyrosyl adenylate and tyrosinyl adenylate bind in similar conformations within a deep cleft in the α β domain. The tyrosine moiety is in the equivalent position to that occupied by tyrosine in crystals of the truncated mutant and makes similar strong polar interactions with the enzyme. The α-phosphate group interacts with the main-chain nitrogen of Asp38. The two hydroxyl groups of the ribose form strong interactions with the protein. The 2′-hydroxyl group interacts with the carboxylate of Asp194 and the main-chain nitrogen of Gly192 while the 3′-hydroxyl interacts with a tightly bound water molecule (Wat326). The adenine moiety appears to make no significant polar interactions with the protein. The results of site-directed mutagenesis studies are examined in the light of these refined structures.

Crystal Structure of the Complex of Ribulose-1,5-bisphosphate Carboxylase and a Transition State Analogue, 2-Carboxy-D-arabinitol 1,5-Bisphosphate

The crystal structure of the binary complex of nonactivated ribulose-1,5-bisphosphate carboxylase/oxygenase from Rhodospirillum rubrum and a transition state analogue, 2-carboxy-D-arabinitol 1,5-bisphosphate has been determined to 2.6 A resolution with x-ray crystallographic methods. The transition state analogue binds in a rather extended conformation at the active site. The orientation of the transition state analogue within the active site could be determined from the electron density maps. The P1 phosphate group of the analogue binds at a site built up of residues from loops 5 and 6 of the alpha/beta-barrel. The phosphate group interacts with the side chains of the conserved residues Arg-288, His-321, and Ser-368 and with main chain nitrogens from residues Thr-322 and Gly-323. The second phosphate group of the transition state analogue binds at the opposite side of the barrel close to loops 1 and 8. Significant differences for the positions and interactions of the P2 phosphate group with the enzyme are found in the two subunits of the dimer. The different mode of binding for this phosphate group in the two subunits is interpreted as a consequence of different conformations of the polypeptide chain observed in loops 6 and 8. The P2 phosphate group interacts with the sidechains of Lys-166 and Lys-329. Loop 6, which is disordered in the nonactivated, nonliganded enzyme is considerably more ordered in one of the subunits, probably due to the interaction of the side chain of Lys-329 with the P2 phosphate group. Almost all oxygen atoms are hydrogen bonded to groups on the enzyme. The carboxyl group forms hydrogen bonds to the side chain of the conserved Asn-111. The binding of the transition state analogue to the nonactivated enzyme is different from the binding of the analogue to activated spinach ribulose-bisphosphate carboxylase.

Crystallographic investigations of nicotinamide adenine dinucleotide binding to horse liver alcohol dehydrogenase.

The binding of NAD to liver alcohol dehydrogenase has been studied in four different ternary complexes by using crystallographic methods. These complexes crystallize isomorphously in a triclinic crystal form which contains the whole dimer of the enzyme in the asymmetric unit. This form of the enzyme has been refined at 2.9-A resolution to a crystallographic R factor of 0.22. NAD binds in essentially the same way in these complexes. The binding site is located at the central part of the coenzyme binding domain. The adenine ring binds with hydrophobic interactions between two isoleucine side chains. Both ribose rings have 2E(C2'-endo) puckering, and each ribose makes three hydrogen bonds to the enzyme. The pyrophosphate bridge has hydrogen bonds to the side chains of arginine-47 and -369 and to main chain nitrogen atoms from the amino ends of two alpha-helices. The nicotinamide ring is in van der Waals contact with the active-site zinc atom and with the sulfur atoms of its cysteine ligands. The carboxamide group is about 30 degrees out of the plane of the nicotinamide ring and hydrogen bonds to main chain atoms of residues 292,317, and 319. The overall conformation of the NAD molecule is similar to that observed for other dehydrogenases, but differs in details. In the presence of the coenzyme, the enzyme undergoes a large conformational change from an open to a closed form. This conformational change has three major effects: to create favorable binding interactions with groups of the enzyme, to enclose the coenzyme and gain binding energy for the coenzyme by reducing the accessible surface area, and to close off one entrance to the active site. As a comparison, ADP-ribose binding has been studied in the open form of the enzyme. The adenosine moiety binds in a similar way as NAD, while the rest of the molecule has different interactions.

Water structure in a protein crystal: Rubredoxin at 1.2 Å resolution

The model for rubredoxin based on X-ray diffraction data has been extensively refined with a 1.2 Å resolution data set. Water oxygen atoms were deleted from the model if B exceeded 50 Å 2 and occupancy was less than 0.3 eÅ −3. The final water model consists of 127 sites with B values ranging from 15 to 6 ̃ 0 Å 2 and occupancies from unity down to 0.3, the most tightly bound water oxygen atoms being hydrogen bonded to two or more main-chain nitrogen or oxygen atoms. The water forms extensive hydrogen bond networks bridging the crevices on the molecular surfaces and between adjacent molecules. The minimum distances of the water sites from the protein surface are distributed about two distinct maxima, the major one at 2.5 to 3 Å and a minor one at 4 to 4.5 Å. Beyond 5 ̃ to 6 Å from the protein surface, the discrete water merges into the aqueous continuum.