The complex of Bacillus pasteurii urease with acetohydroxamate anion from X-ray data at 1.55 Å resolution

The structure of Bacillus pasteurii urease inhibited with acetohydroxamic acid was solved and refined anisotropically using synchrotron X-ray cryogenic diffraction data (1.55 A resolution, 99.5% completeness, data redundancy = 26, R-factor = 15.1%, PDB code 4UBP). The two Ni ions in the active site are separated by a distance of 3.53 A. The structure clearly shows the binding mode of the inhibitor anion, symmetrically bridging the two Ni ions in the active site through the hydroxamate oxygen and chelating one Ni ion through the carbonyl oxygen. The flexible flap flanking the active site cavity is in the open conformation. The possible implications of the results on structure-based molecular design of new urease inhibitors are discussed.     

A molecular mechanical analysis of the active site of urease with a special emphasis on determining the binding conformations available to oxygen-bound urea.

In order to model the active site of urease which contains two nickel ions with differing coordination geometries new parameters were derived for the AMBER* force field. These parameters were obtained by structure based optimization and use a single set of parameters with points on a sphere approach to model nickel(II) high-spin in all its coordination geometries. The force field was successfully used to model the active site of urease and to predict that a bridging water between the two nickel ions in urease was missing from the solid state structure of urease. A thorough conformational search was undertaken to find the conformations available to urea within urease. All the low energy conformations found were used to determine a consensus urea binding model.     

Prediction of interaction sites from apo 3D structures when the holo conformation is different

The predictability of catalytic and binding sites from apo structures is addressed for proteins that undergo significant conformational change upon binding. Theoretical microscopic titration curves (THEMATICS), an electrostatics-based method for the prediction of functional sites, is performed on a test set of 24 proteins with both apo and holo structures available. For 23 of these 24 proteins (96%), THEMATICS predicts the correct catalytic or binding site for both the apo and holo forms. For only one of the 24 proteins, THEMATICS makes the correct prediction for the holo structure but fails for the apo structure. The metrics used by THEMATICS to identify functional residues generally are larger in absolute value for the functional residues in the holo forms compared to the corresponding residues in the apo forms. However, even in the apo forms, these identifying metrics are still statistically significantly larger for functional residues than for residues not involved in catalysis or binding. This indicates that some of the unusual electrostatic properties of functional residues are preserved in the apo conformation. Evidence is presented that certain residues immediately surrounding the active catalytic and binding residues impart functionally important chemical and electrostatic properties to the active residues. At least parts of these microenvironments exist in the unbound conformations, such that THEMATICS is able to distinguish the functional residues even in the apo structures.     

超氧化物歧化酶全酶和脱辅基酶胍变性时失活与去折叠的比较研究

利用紫外差吸收光谱和荧光发射光谱等监测手段研究天然铜锌ＳＯＤ（ｈｏｌｏ－ＳＯＤ）和脱铜锌ＳＯＤ（ａｐｏ－ＳＯＤ）在不同浓度胍溶液中的去折叠及活力变化．结果表明ｈｏｌｏ－ＳＯＤ和ａｐｏ－ＳＯＤ分别在４．０和２．０ｍｏｌ／Ｌ胍溶液中去折叠,而分别在２．０和０．５ｍｏｌ／Ｌ胍溶液中其构象尚未发生明显改变时活性几乎完全丧失．提示金属离子对维持酶的整体及活性部位构象具有重要作用,脱去金属离子的酶分子的构象特别是活性部位的构象更易受到变性剂的破坏．     

The complex of Bacillus pasteurii urease with β-mercaptoethanol from X-ray data at 1.65-Å resolution

The structure of β-mercaptoethanol-inhibited urease from Bacillus pasteurii, a highly ureolytic soil micro-organism, was solved at 1.65?Å using synchrotron X-ray cryogenic diffraction data. The structure clearly shows the unexpected binding mode of β-mercaptoethanol, which bridges the two nickel ions in the active site through the sulfur atom and chelates one Ni through the OH functionality. Another molecule of inhibitor forms a mixed disulfide with a Cys residue, thus sealing the entrance to the active site cavity by steric hindrance. The possible implications of the results on structure-based molecular design of new urease inhibitors are discussed.     

Crystal structure of the apo form of a β‐transaminase from Mesorhizobium sp. strain LUK

Pyridoxal 5′‐phosphate (PLP)‐dependent β‐transaminases (βTAs) reversibly catalyze transamination reactions by recognizing amino groups linked to the β‐carbon atoms of their substrates. Although several βTA structures have been determined as holo forms containing PLP, little is known about the effect of PLP on the conversion of the apo structure to the holo structure. We determined the crystal structure of the apo form of a βTA from Mesorhizobium sp. strain LUK at 2.2 Å resolution to elucidate how PLP affects the βTA structure. The structure revealed three major disordered regions near the active site. Structural comparison with the holo form also showed that the disordered regions in the apo form are ordered and partially adopt secondary structures in the holo form. These findings suggest that PLP incorporation into the active site contributes to the structural stability of the active site architecture, thereby forming the complete active site. Our results provide novel structural insights into the role of PLP in terms of active site formation.     

Effects of Zn(II) binding and apoprotein structural stability on the conformation change of designed antennafinger proteins

Ligand-induced conformation change is a general strategy for controlling protein function. In this work, we demonstrate the relationships between ligand binding and conformational stability using a previously designed protein, Ant-F, which undergoes a conformation change upon Zn(II) binding. To investigate the effect of stabilization of the apo structure on the conformation change, we also created a novel protein, Ant-F-H1, into which mutations are introduced to increase its stability over that of Ant-F. The chemical denaturation experiments clarified that apo-Ant-F-H1 is more stable than apo-Ant-F (DeltaDeltaG = -1.28 kcal/mol) and that the stability of holo-Ant-F-H1 is almost the same as that of holo-Ant-F. The Zn(II) binding assay shows that the affinity of Zn(II) for Ant-F-H1 is weaker than that for Ant-F (DeltaDeltaG = 1.40 kcal/mol). A large part of the increased value of free energy in stability corresponds to the decreased value of free energy in Zn(II) binding, indicating that the stability of the apo structure directly affects the conformation change. The denaturation experiments also reveal that Zn(II) destabilizes the conformation of both proteins. From the thermodynamic linkage, Zn(II) is thought to bind to the unfolded state with high affinity. These results suggest that the binding of Zn(II) to the unfolded state is an important factor in the conformational change as well as the stability of the apo and holo structures.     

Molecular dynamics simulations reveal that apo‐HisJ can sample a closed conformation

The Escherichia coli histidine binding protein HisJ is a type II periplasmic binding protein (PBP) that preferentially binds histidine and interacts with its cytoplasmic membrane ABC transporter, HisQMP₂, to initiate histidine transport. HisJ is a bilobal protein where the larger Domain 1 is connected to the smaller Domain 2 via two linking strands. Type II PBPs are thought to undergo “Venus flytrap” movements where the protein is able to reversibly transition from an open to a closed conformation. To explore the accessibility of the closed conformation to the apo state of the protein, we performed a set of all‐atom molecular dynamics simulations of HisJ starting from four different conformations: apo‐open, apo‐closed, apo‐semiopen, and holo‐closed. The simulations reveal that the closed conformation is less dynamic than the open one. HisJ experienced closing motions and explored semiopen conformations that reverted to closed forms resembling those found in the holo‐closed state. Essential dynamics analysis of the simulations identified domain closing/opening and twisting as main motions. The formation of specific inter‐hinge strand and interdomain polar interactions contributed to the adoption of the closed apo‐conformations although they are up to 2.5‐fold less prevalent compared with the holo‐closed simulations. The overall sampling of the closed form by apo‐HisJ provides a rationale for the binding of unliganded PBPs with their cytoplasmic membrane ABC transporters. Proteins 2014; 82:386–398. © 2013 Wiley Periodicals, Inc.     

Effect of metal ion binding on the secondary structure of bovine alpha-lactalbumin as examined by infrared spectroscopy

We have examined the influence of monovalent and divalent cations on the secondary structure of bovine alpha-lactalbumin at neutral pH using Fourier-transform infrared spectroscopy. Our present studies are based on previously reported amide I' component band assignments for this protein [Prestrelski, S. J., Byler, D. M., & Thompson, M. P. (1991) Int. J. Pept. Protein Res. 37, 508-512]. The results indicate that upon dissolution, alpha-lactalbumin undergoes a small, but significant, time-dependent conformational change, regardless of the ions present. Additionally, these studies provide the first quantitative measure of the well-known secondary structural change which accompanies calcium binding. Results indicate that removal of Ca2+ from holo alpha-lactalbumin results in local unfolding of the Ca(2+)-binding loop; the spectra indicate that approximately 16% of the backbone chain changes from a rigid coordination complex to an unordered loop. We have also examined the effects of binding of several other metal ions. Our studies have revealed that binding of Mn2+ to apo alpha-lactalbumin (Ca(2+)-free), while inducing a small, but significant, conformational change, does not cause the alpha-lactalbumin backbone conformation to change to that of the holo (Ca(2+)-bound) form as characterized by infrared spectroscopy. Similar changes to those induced by Mn2+ are observed upon binding of Na+ to apo alpha-lactalbumin, and furthermore, even at very high concentrations (0.2 M), Na+ does not stabilize a structure similar to the holo form. Binding of Zn2+ to the apo form of alpha-lactalbumin does not result in significant backbone conformational changes, suggesting a rigid Zn(2+)-binding site.(ABSTRACT TRUNCATED AT 250 WORDS)     

Molecular Basis of Calcium-Sensitizing and Desensitizing Mutations of the Human Cardiac Troponin C Regulatory Domain: A Multi-Scale Simulation Study

Troponin C (TnC) is implicated in the initiation of myocyte contraction via binding of cytosolic and subsequent recognition of the Troponin I switch peptide. Mutations of the cardiac TnC N-terminal regulatory domain have been shown to alter both calcium binding and myofilament force generation. We have performed molecular dynamics simulations of engineered TnC variants that increase or decrease sensitivity, in order to understand the structural basis of their impact on TnC function. We will use the distinction for mutants that are associated with increased affinity and for those mutants with reduced affinity. Our studies demonstrate that for GOF mutants V44Q and L48Q, the structure of the physiologically-active site II binding site in the -free (apo) state closely resembled the -bound (holo) state. In contrast, site II is very labile for LOF mutants E40A and V79Q in the apo form and bears little resemblance with the holo conformation. We hypothesize that these phenomena contribute to the increased association rate, , for the GOF mutants relative to LOF. Furthermore, we observe significant positive and negative positional correlations between helices in the GOF holo mutants that are not found in the LOF mutants. We anticipate these correlations may contribute either directly to affinity or indirectly through TnI association. Our observations based on the structure and dynamics of mutant TnC provide rationale for binding trends observed in GOF and LOF mutants and will guide the development of inotropic drugs that target TnC.     

Structure-based rationalization of urease inhibition by phosphate: novel insights into the enzyme mechanism

The structure of Bacillus pasteurii urease (BPU) inhibited with phosphate was solved and refined using synchrotron X-ray diffraction data from a vitrified crystal (1.85 A resolution, 99.3% completeness, data redundancy 4.6, R-factor 17.3%, PDB code 6UBP). A distance of 3.5 A separates the two Ni ions in the active site. The binding mode of the inhibitor involves the formation of four coordination bonds with the two Ni ions: one phosphate oxygen atom symmetrically bridges the two metal ions (1.9-2.0 A), while two of the remaining phosphate oxygen atoms bind to the Ni atoms at 2.4 A. The fourth phosphate oxygen is directed into the active site channel. Analysis of the H-bonding network around the bound inhibitor indicates that phosphate is bound as the H2PO4- anion, and that an additional proton is present on the Odelta2 atom of Asp(alpha363), an active site residue involved in Ni coordination through Odelta1. The flexible flap flanking the active site cavity is in the open conformation. Analysis of the complex reveals why phosphate is a relatively weak inhibitor and why sulfate does not bind to the nickels in the active site. The implications of the results for the understanding of the urease catalytic mechanism are reviewed. A novel alternative for the proton donor is presented.     

Smyd3 open & closed lock mechanism for substrate recruitment: The hinge motion of C-terminal domain inferred from μ-second molecular dynamics simulations

BackgroundThe human lysine methyltransferase Smyd3, a member of the SET and MYND domain containing protein family, harbors methylation activity on both histone and non-histone targets in a tightly regulated manner. The mechanism of how Smyd3 dynamically regulates substrate recognition is still not fully unveiled.MethodsHere, we employed molecular dynamics simulations on full length human Smyd3, performed to a total of 1.2 μ-second, in the presence (holo) and absence (apo) of the S-Adenosyl methionine (AdoMet) cofactor. The dynamical features of Smyd3 in apo and holo states have been examined and compared via examining geometrical and electrostatic properties.ResultsThe results show a distinct dynamics of the C-terminal domain (CTD) in the two states. In the apo state, the CTD undergoes a large hinge like motion and samples more opened configurations, thus acting like a loosened clamp and resulting in expanded substrate binding crevice. In the holo state, the CTD exhibits a restricted motion while the overall structure remains compact, mimicking a closed clamp. This leads to a localized increase in the negative potential at the substrate binding cleft. Further, solvent accessibility of critical residues at the target lysine access channel, important for methylation activity, is increased.ConclusionsWe postulate that AdoMet cofactor acts like a key and locks Smyd3 in a closed conformation. In effect, the cofactor binding restricts the elasticity of the CTD, presenting a compact substrate binding cleft with high negative potential, which may have implications on substrate recruitment via long range electrostatics.General significanceThe deletion of the CTD from Smyd3 has been shown to abolish the basal histone methylation activity. Our study highlights the importance of the CTD elasticity in shaping the substrate binding site for recognition and supports the previously proposed role of the CTD in stabilizing the active site for methylation activity.     

Prediction of transition metal-binding sites from apo protein structures

Metal ions are crucial for protein function. They participate in enzyme catalysis, play regulatory roles, and help maintain protein structure. Current tools for predicting metal-protein interactions are based on proteins crystallized with their metal ions present (holo forms). However, a majority of resolved structures are free of metal ions (apo forms). Moreover, metal binding is a dynamic process, often involving conformational rearrangement of the binding pocket. Thus, effective predictions need to be based on the structure of the apo state. Here, we report an approach that identifies transition metal-binding sites in apo forms with a resulting selectivity >95%. Applying the approach to apo forms in the Protein Data Bank and structural genomics initiative identifies a large number of previously unknown, putative metal-binding sites, and their amino acid residues, in some cases providing a first clue to the function of the protein.     

A new proposal for urease mechanism based on the crystal structures of the native and inhibited enzyme from Bacillus pasteurii: why urea hydrolysis costs two nickels.

BACKGROUND: Urease catalyzes the hydrolysis of urea, the final step of organic nitrogen mineralization, using a bimetallic nickel centre. The role of the active site metal ions and amino acid residues has not been elucidated to date. Many pathologies are associated with the activity of ureolytic bacteria, and the efficiency of soil nitrogen fertilization with urea is severely decreased by urease activity. Therefore, the development of urease inhibitors would lead to a reduction of environmental pollution, to enhanced efficiency of nitrogen uptake by plants, and to improved therapeutic strategies for treatment of infections due to ureolytic bacteria. Structure-based design of urease inhibitors would require knowledge of the enzyme mechanism at the molecular level. RESULTS: The structures of native and inhibited urease from Bacillus pasteurii have been determined at a resolution of 2.0 A by synchrotron X-ray cryogenic crystallography. In the native enzyme, the coordination sphere of each of the two nickel ions is completed by a water molecule and a bridging hydroxide. A fourth water molecule completes a tetrahedral cluster of solvent molecules. The enzyme crystallized in the presence of phenylphosphorodiamidate contains the tetrahedral transition-state analogue diamidophosphoric acid, bound to the two nickel ions in an unprecedented mode. Comparison of the native and inhibited structures reveals two distinct conformations of the flap lining the active-site cavity. CONCLUSIONS: The mode of binding of the inhibitor, and a comparison between the native and inhibited urease structures, indicate a novel mechanism for enzymatic urea hydrolysis which reconciles the available structural and biochemical data.     

Closed conformation of the active site loop of rabbit muscle triosephosphate isomerase in the absence of substrate: evidence of conformational heterogeneity

The active site loop of triosephosphate isomerase (TIM) exhibits a hinged-lid motion, alternating between the two well defined "open" and "closed" conformations. Until now the closed conformation had only been observed in protein complexes with substrate analogues. Here, we present the first rabbit muscle apo TIM structure, refined to 1.5A resolution, in which the active site loop is either in the open or in the closed conformation in different subunits of the enzyme. In the closed conformation described here, the lid loop residues participate in stabilizing hydrogen bonds characteristic of holo TIM structures, whereas chemical interactions observed in the open loop conformation are similar to those found in the apo structures of TIM. In the closed conformation, a number of water molecules are observed at the projected ligand atom positions that are hydrogen bonded to the active site residues. Additives used during crystallization (DMSO and Tris molecules and magnesium atoms) were modeled in the electron density maps. However, no specific binding of these molecules is observed at, or close to, the active site and the lid loop. To further investigate this unusual closed conformation of the apo enzyme, two more rabbit muscle TIM structures, one in the same and another in a different crystal form, were determined. These structures present the open lid conformation only, indicating that the closed conformation cannot be explained by crystal contact effects. To rationalize why the active site loop is closed in the absence of ligand in one of the subunits, extensive comparison with previously solved TIM structures was carried out, supported by the bulk of available experimental information about enzyme kinetics and reaction mechanism of TIM. The observation of both open and closed lid conformations in TIM crystals might be related to a persistent conformational heterogeneity of this protein in solution.     

Biliverdin Reductase B Dynamics Are Coupled to Coenzyme Binding

Biliverdin reductase B (BLVRB) is a newly identified cellular redox regulator that catalyzes the NADPH-dependent reduction of multiple substrates. Through mass spectrometry analysis, we identified high levels of BLVRB in mature red blood cells, highlighting the importance of BLVRB in redox regulation. The BLVRB conformational changes that occur during conezyme/substrate binding and the role of dynamics in BLVRB function, however, remain unknown. Through a combination of NMR, kinetics, and isothermal titration calorimetry studies, we determined that BLVRB binds its coenzyme 500-fold more tightly than its substrate. While the active site of apo BLVRB is highly dynamic on multiple timescales, active site dynamics are largely quenched within holo BLVRB, in which dynamics are redistributed to other regions of the enzyme. We show that a single point mutation of Arg78?Ala leads to both an increase in active site micro-millisecond motions and an increase in the microscopic rate constants of coenzyme binding. This demonstrates that altering BLVRB active site dynamics can directly cause a change in functional characteristics. Our studies thus address the solution behavior of apo and holo BLVRB and identify a role of enzyme dynamics in coenzyme binding.     

Conformational selection in silico: loop latching motions and ligand binding in enzymes

Ligand binding frequently induces significant conformational changes in a protein receptor. Understanding and predicting such conformational changes represent an important challenge for computational biology, including applications to structure-based drug design. We describe an approach to this problem based on the assumption that the holo state is at least transiently populated in the absence of a ligand; this hypothesis has been referred to as "conformational selection." Here, we apply a method that tests this hypothesis on a challenging class of ligand-induced conformational changes, which we refer to as loop latching: the closing of a loop around an active site that sequesters the ligand from solvent. The method uses a combination of replica exchange molecular dynamics and a loop prediction algorithm to generate low-energy loop structures, and docking to select the conformation appropriate for binding a particular ligand. On a test set of six proteins, it yields loop structures including hololike conformations, generally below 2 A RMSD from the liganded structure, for loops that span up to 15 residues. Docking serves as a stringent test of the predictions. In five of the six cases, the predicted loop conformations improve the ranks of cognate ligands relative to using the apo structure, although the results remain, in most cases, significantly worse than using a holo structure. The poses of the cognate ligands are correct in four of the six test cases, while they are correct for five of the six using a holo structure.     

Jack bean urease: the effect of active-site binding inhibitors on the reactivity of enzyme thiol groups

In view of the complexity of the role of the active site flap cysteine in the urease catalysis, in this work we studied how the presence of typical active-site binding inhibitors of urease, phenylphosphorodiamidate (PPD), acetohydroxamic acid (AHA), boric acid and fluoride, affects the reactivity of enzyme thiol groups, the active site flap thiol in particular. For that the inhibitor-urease complexes were prepared with excess inhibitors and had their thiol groups titrated with DTNB. The effects observed were analyzed in terms of the structures of the inhibitor-urease complexes reported in the literature. We found that the effectiveness in preventing the active site cysteine from the modification by disulfides, varied among the inhibitors studied, even though they all bind to the active site. The variations were accounted for by different extents of geometrical distortion in the active site that the inhibitors introduced upon binding, leaving the flap either open in AHA-, boric acid- and fluoride-inhibited urease, like in the native enzyme or closed in PPD-inhibited urease. Among the inhibitors, only PPD was found to be able to thoroughly protect the flap cysteines from the further reaction with disulfides, this apparently resulting from the closed conformation of the flap. Accordingly, in practical terms PPD may be regarded as the most suitable inhibitor for active-site protection experiments in inhibition studies of urease.     

Structure of UreG/UreF/UreH Complex Reveals How Urease Accessory Proteins Facilitate Maturation of Helicobacter pylori Urease

Urease is a metalloenzyme essential for the survival of Helicobacter pylori in acidic gastric environment. Maturation of urease involves carbamylation of Lys219 and insertion of two nickel ions at its active site. This process requires GTP hydrolysis and the formation of a preactivation complex consisting of apo-urease and urease accessory proteins UreF, UreH, and UreG. UreF and UreH form a complex to recruit UreG, which is a SIMIBI class GTPase, to the preactivation complex. We report here the crystal structure of the UreG/UreF/UreH complex, which illustrates how UreF and UreH facilitate dimerization of UreG, and assembles its metal binding site by juxtaposing two invariant Cys66-Pro67-His68 metal binding motif at the interface to form the (UreG/UreF/UreH)₂ complex. Interaction studies revealed that addition of nickel and GTP to the UreG/UreF/UreH complex releases a UreG dimer that binds a nickel ion at the dimeric interface. Substitution of Cys66 and His68 with alanine abolishes the formation of the nickel-charged UreG dimer. This nickel-charged UreG dimer can activate urease in vitro in the presence of the UreF/UreH complex. Static light scattering and atomic absorption spectroscopy measurements demonstrated that the nickel-charged UreG dimer, upon GTP hydrolysis, reverts to its monomeric form and releases nickel to urease. Based on our results, we propose a mechanism on how urease accessory proteins facilitate maturation of urease.     

Crystal structures of the Chromobacterium violaceumω-transaminase reveal major structural rearrangements upon binding of coenzyme PLP

The bacterial ω-transaminase from Chromobacterium violaceum (Cv-ωTA, EC2.6.1.18) catalyses industrially important transamination reactions by use of the coenzyme pyridoxal 5'-phosphate (PLP). Here, we present four crystal structures of Cv-ωTA: two in the apo form, one in the holo form and one in an intermediate state, at resolutions between 1.35 and 2.4 ?. The enzyme is a homodimer with a molecular mass of ～ 100 kDa. Each monomer has an active site at the dimeric interface that involves amino acid residues from both subunits. The apo-Cv-ωTA structure reveals unique 'relaxed' conformations of three critical loops involved in structuring the active site that have not previously been seen in a transaminase. Analysis of the four crystal structures reveals major structural rearrangements involving elements of the large and small domains of both monomers that reorganize the active site in the presence of PLP. The conformational change appears to be triggered by binding of the phosphate group of PLP. Furthermore, one of the apo structures shows a disordered 'roof?' over the PLP-binding site, whereas in the other apo form and the holo form the 'roof' is ordered. Comparison with other known transaminase crystal structures suggests that ordering of the 'roof' structure may be associated with substrate binding in Cv-ωTA and some other transaminases. DATABASE: The atomic coordinates and structure factors for the Chromobacterium violaceumω-transaminase crystal structures can be found in the RCSB Protein Data Bank (http://www.rcsb.org) under the accession codes 4A6U for the holoenzyme, 4A6R for the apo1 form, 4A6T for the apo2 form and 4A72 for the mixed form STRUCTURED DIGITAL ABSTRACT: ? -transaminases and -transaminases bind by dynamic light scattering (View interaction) ? -transaminase and -transaminase bind by x-ray crystallography (View interaction) ? -transaminase and -transaminase bind by x-ray crystallography (View interaction).