Conservation of Flexible Residue Clusters among Structural and Functional Enzyme Homologues

Conformational flexibility between structural ensembles is an essential component of enzyme function. Although the broad dynamical landscape of proteins is known to promote a number of functional events on multiple time scales, it is yet unknown whether structural and functional enzyme homologues rely on the same concerted residue motions to perform their catalytic function. It is hypothesized that networks of contiguous and flexible residue motions occurring on the biologically relevant millisecond time scale evolved to promote and/or preserve optimal enzyme catalysis. In this study, we use a combination of NMR relaxation dispersion, model-free analysis, and ligand titration experiments to successfully capture and compare the role of conformational flexibility between two structural homologues of the pancreatic ribonuclease family: RNase A and eosinophil cationic protein (or RNase 3). In addition to conserving the same catalytic residues and structural fold, both homologues show similar yet functionally distinct clusters of millisecond dynamics, suggesting that conformational flexibility can be conserved among analogous protein folds displaying low sequence identity. Our work shows that the reduced conformational flexibility of eosinophil cationic protein can be dynamically and functionally reproduced in the RNase A scaffold upon creation of a chimeric hybrid between the two proteins. These results support the hypothesis that conformational flexibility is partly required for catalytic function in homologous enzyme folds, further highlighting the importance of dynamic residue sectors in the structural organization of proteins.     

Mutagenesis of residues flanking Lys-40 enhances the enzymatic activity and reduces the angiogenic potency of angiogenin.

The primary structure of the blood vessel inducing protein angiogenin is 35% identical with that of pancreatic ribonuclease (RNase) and contains counterparts for the critical RNase active-site residues His-12, Lys-41, and His-119. Although angiogenin is a ribonucleolytic enzyme, its activity toward conventional substrates is lower than that of pancreatic RNase by several orders of magnitude. Comparison of the amino acid sequences of RNase and angiogenin reveals several striking differences in the region flanking the active-site lysine, including a deletion and a transposition of aspartic acid and proline residues. In order to examine how these sequence changes alter the functional properties of angiogenin, an angiogenin/RNase hybrid protein (ARH-II), in which residues 38-41 of angiogenin (Pro-Cys-Lys-Asp) have been replaced by the corresponding segment of bovine pancreatic RNase (Asp-Arg-Cys-Lys-Pro), was prepared by regional mutagenesis. Compared to angiogenin, ARH-II has markedly diminished angiogenic activity on the chick embryo chorioallantoic membrane but 5-75-fold greater enzymatic activity toward a variety of polynucleotide and dinucleotide substrates. In addition, the specificity of ARH-II toward dinucleotide substrates differs from that of angiogenin and is qualitatively similar to that of pancreatic RNase. Thus, non-active-site residues near Lys-40 in angiogenin appear to play a significant role in determining enzymatic specificity and reactivity as well as angiogenic potency. An additional angiogenin/RNase hybrid protein (ARH-IV), in which residues 59-71 of ARH-II have been replaced by the corresponding segment of pancreatic RNase, was also prepared.(ABSTRACT TRUNCATED AT 250 WORDS)     

Crystallographic studies on structural features that determine the enzymatic specificity and potency of human angiogenin: Thr44, Thr80, and residues 38-41

Human angiogenin (Ang) is a potent inducer of blood vessel formation and is a member of the pancreatic ribonuclease superfamily. Its enzymatic activity is unusually weak and biased toward cleavage after cytidine nucleotides. As part of an ongoing investigation into the structural basis of Ang's characteristic activity, we have determined the crystal structures of three Ang variants having novel activity. (i) The structure of T44D-Ang indicates that Asp44 can participate directly in pyrimidine binding and that the intrinsic hydrogen-bonding capability of this residue largely governs the pyrimidine specificity of this variant. Unexpectedly, the mutation also causes the most extensive disruption of the C-terminus seen in any Ang variant thus far. This allows the side chain of Arg101 to penetrate the B(1) site, raising the possibility that it participates in substrate binding as occurs in ribonuclease 4. (ii) The structure of T80A-Ang supports the view that Thr80 plays little role in maintaining the obstructive conformation of the C-terminus and that its participation in a hydrogen bond with Thr44 selectively weakens the interaction between Thr44 and N3 of cytosine. (iii) ARH-II is an angiogenin/RNase A chimera in which residues 38-41 of Ang are replaced with the corresponding residues (38-42) of RNase A. Its structure suggests that the guest segment influences catalysis by subtle means, possibly by reducing the pK(a) of the catalytic lysine. The loss of angiogenic activity is not attributable to disruption of known cell-binding or nuclear translocation sites but may be a consequence of the chimera's enhanced ribonucleolytic activity.     

Amino acid sequence of the nonsecretory ribonuclease of human urine

The amino acid sequence of a nonsecretory ribonuclease isolated from human urine was determined except for the identity of the residue at position 7. Sequence information indicates that the ribonucleases of human liver and spleen and an eosinophil-derived neurotoxin are identical or very closely related gene products. The sequence is identical at about 30% of the amino acid positions with those of all of the secreted mammalian ribonucleases for which information is available. Identical residues include active-site residues histidine-12, histidine-119, and lysine-41, other residues known to be important for substrate binding and catalytic activity, and all eight half-cystine residues common to these enzymes. Major differences include a deletion of six residues in the (so-called) S-peptide loop, insertions of two, and nine residues, respectively, in three other external loops of the molecule, and an addition of three residues at the amino terminus. The sequence shows the human nonsecretory ribonuclease to belong to the same ribonuclease superfamily as the mammalian secretory ribonucleases, turtle pancreatic ribonuclease, and human angiogenin. Sequence data suggest that a gene duplication occurred in an ancient vertebrate ancestor; one branch led to the nonsecretory ribonuclease, while the other branch led to a second duplication, with one line leading to the secretory ribonucleases (in mammals) and the second line leading to pancreatic ribonuclease in turtle and an angiogenic factor in mammals (human angiogenin). The nonsecretory ribonuclease has five short carbohydrate chains attached via asparagine residues at the surface of the molecule; these chains may have been shortened by exoglycosidase action.(ABSTRACT TRUNCATED AT 250 WORDS)     

Active-site residues move independently from the rest of the protein in a 200 ns molecular dynamics simulation of cytochrome P450 CYP119

The conformational dynamics of cytochrome P450 enzymes are critical to their catalytic activity. In this study, the correlated motion between residues in a 200 ns molecular dynamics trajectory of the thermophilic CYP119 was analyzed to parse out conformational relationships. Residues that are structurally related, for example residues within a helix, generally have highly correlated motion. In addition, clusters of non-adjacent residues that show correlated motion (“hot spots”) are seen in various regions, including at the base of the F and G helices that make up the most dynamic region of the enzyme. A modified k-means algorithm that clusters residues based on their correlated motion indicates that functionally related residues are in the same cluster (e.g., the catalytic threonines and the heme). Tightly coupled clusters form a solvent-exposed “shell” around the enzyme, whereas less coupling between clusters is seen in regions that are critical to ligand interactions, redox partner interactions, and catalysis. Most notably, we find that residues in the active site move independently from the rest of the enzyme, effectively insulating the catalytic machinery from other regions of the protein.     

Alteration of hydrogen bonding in the vicinity of histidine 48 disrupts millisecond motions in RNase A

The motion of amino acid residues on the millisecond (ms) time scale is involved in the tight regulation of catalytic function in numerous enzyme systems. Using a combination of mutational, enzymological, and relaxation-compensated (15)N Carr-Purcell-Meiboom-Gill (CPMG) methods, we have previously established the conformational significance of the distant His48 residue and the neighboring loop 1 in RNase A function. These studies suggested that RNase A relies on an intricate network of hydrogen bonding interactions involved in propagating functionally relevant, long-range ms motions to the catalytic site of the enzyme. To further investigate the dynamic importance of this H-bonding network, this study focuses on the individual replacement of Thr17 and Thr82 with alanine, effectively altering the key H-bonding interactions that connect loop 1 and His48 to the rest of the protein. (15)N CPMG dispersion studies, nuclear magnetic resonance (NMR) chemical shift analysis, and NMR line shape analysis of point mutants T17A and T82A demonstrate that the evolutionarily conserved single H-bond linking His48 to Thr82 is essential for propagating ms motions from His48 to the active site of RNase A on the time scale of catalytic turnover, whereas the T17A mutation increases the off rate and conformational exchange motions in loop 1. Accumulating evidence from our mutational studies indicates that residues experiencing conformational exchange in RNase A can be grouped into two separate clusters displaying distinct dynamical features, which appear to be independently affected by mutation. Overall, this study illuminates how tightly controlled and finely tuned ms motions are in RNase A, suggesting that designed modulation of protein motions may be possible.     

Angiogenin and Its Functions in Angiogenesis

The review is devoted to angiogenin, one of the factors that induce formation of blood vessels, which is unique in that it is a ribonuclease. Consideration is given to the tertiary structure of human angiogenin; the catalytic and cell receptor binding sites, their significance for angiogenic activity; the human angiogenin gene structure, chromosomal localization, and expression; the specificity of angiogenin as a ribonuclease and abolishment of protein synthesis; the nuclear localization of angiogenin in proliferating endothelial cells and its significance for angiogenic activity; angiogenin binding to cell surface actin as a plausible mechanism of inducing neovascularization (enhancement of plasminogen activation by actin, stimulation of the cell-associated proteolytic activity; promotion of the cultured cell invasiveness); modulation of mitogenic stimuli in endothelial, smooth muscle, and fibroblast cells by angiogenin. The importance of angiogenin as an adhesive molecule for endothelial and tumor cells is discussed too, as well as the modulation of tubular morphogenesis by bovine angiogenin, prevention of tumor growth in vivoby angiogenin antagonists, prospects of the use of angiogenin and angiogenin-encoding recombinant plasmids and vaccinia virus in therapeutic practice.     

Mechanisms of loss of functions of human angiogenin variants implicated in amyotrophic lateral sclerosis

Background

Mutations in the coding region of angiogenin (ANG) gene have been found in patients suffering from Amyotrophic Lateral Sclerosis (ALS). Neurodegeneration results from the loss of angiogenic ability of ANG (protein coded by ANG). In this work, we performed extensive molecular dynamics (MD) simulations of wild-type ANG and disease associated ANG variants to elucidate the mechanism behind the loss of ribonucleolytic activity and nuclear translocation activity, functions needed for angiogenesis.

Methodology/Principal Findings

MD simulations were carried out to study the structural and dynamic differences in the catalytic site and nuclear localization signal residues between WT-ANG (Wild-type ANG) and six mutants. Variants K17I, S28N, P112L and V113I have confirmed association with ALS, while T195C and A238G single nucleotide polymorphisms (SNPs) encoding L35P and K60E mutants respectively, have not been associated with ALS. Our results show that loss of ribonucleolytic activity in K17I is caused by conformational switching of the catalytic residue His114 by 99°. The loss of nuclear translocation activity of S28N and P112L is caused by changes in the folding of the residues ³¹RRR³³ that result in the reduction in solvent accessible surface area (SASA). Consequently, we predict that V113I will exhibit loss of angiogenic properties by loss of nuclear translocation activity and L35P by loss of both ribonucleolytic activity and nuclear translocation activity. No functional loss was inferred for K60E. The MD simulation results were supported by hydrogen bond interaction analyses and molecular docking studies.

Conclusions/Significance

Conformational switching of catalytic residue His114 seems to be the mechanism causing loss of ribonucleolytic activity and reduction in SASA of nuclear localization signal residues ³¹RRR³³ results in loss of nuclear translocation activity in ANG mutants. Therefore, we predict that L35P mutant, would exhibit loss of angiogenic functions, and hence would correlate with ALS while K60E would not show any loss.     

Molecular Determinants Underlying Binding Specificities of the ABL Kinase Inhibitors: Combining Alanine Scanning of Binding Hot Spots with Network Analysis of Residue Interactions and Coevolution

Quantifying binding specificity and drug resistance of protein kinase inhibitors is of fundamental importance and remains highly challenging due to complex interplay of structural and thermodynamic factors. In this work, molecular simulations and computational alanine scanning are combined with the network-based approaches to characterize molecular determinants underlying binding specificities of the ABL kinase inhibitors. The proposed theoretical framework unveiled a relationship between ligand binding and inhibitor-mediated changes in the residue interaction networks. By using topological parameters, we have described the organization of the residue interaction networks and networks of coevolving residues in the ABL kinase structures. This analysis has shown that functionally critical regulatory residues can simultaneously embody strong coevolutionary signal and high network centrality with a propensity to be energetic hot spots for drug binding. We have found that selective (Nilotinib) and promiscuous (Bosutinib, Dasatinib) kinase inhibitors can use their energetic hot spots to differentially modulate stability of the residue interaction networks, thus inhibiting or promoting conformational equilibrium between inactive and active states. According to our results, Nilotinib binding may induce a significant network-bridging effect and enhance centrality of the hot spot residues that stabilize structural environment favored by the specific kinase form. In contrast, Bosutinib and Dasatinib can incur modest changes in the residue interaction network in which ligand binding is primarily coupled only with the identity of the gate-keeper residue. These factors may promote structural adaptability of the active kinase states in binding with these promiscuous inhibitors. Our results have related ligand-induced changes in the residue interaction networks with drug resistance effects, showing that network robustness may be compromised by targeted mutations of key mediating residues. This study has outlined mechanisms by which inhibitor binding could modulate resilience and efficiency of allosteric interactions in the kinase structures, while preserving structural topology required for catalytic activity and regulation.     

A dynamic loop provides dual control over the catalytic and membrane binding activity of a bacterial serine hydrolase

The bacterial acyl protein thioesterase (APT) homologue FTT258 from the gram-negative pathogen Francisella tularensis exists in equilibrium between a closed and open state. Interconversion between these two states is dependent on structural rearrangement of a dynamic loop overlapping its active site. The dynamics and structural properties of this loop provide a simple model for how the catalytic activity of FTT258 could be spatiotemporally regulated within the cell. Herein, we characterized the dual roles of this dynamic loop in controlling its catalytic and membrane binding activity. Using a comprehensive library of loop variants, we determined the relative importance of each residue in the loop to these two biological functions. For the catalytic activity, a centrally located tryptophan residue (Trp66) was essential, with the resulting alanine variant showing complete ablation of enzyme activity. Detailed analysis of Trp66 showed that its hydrophobicity in combination with spatial arrangement defined its essential role in catalysis. Substitution of other loop residues congregated along the N-terminal side of the loop also significantly impacted catalytic activity, indicating a critical role for this loop in controlling catalytic activity. For membrane binding, the centrally located hydrophobic residues played a surprising minor role in membrane binding. Instead general electrostatic interactions regulated membrane binding with positively charged residues bracketing the dynamic loop controlling membrane binding. Overall for FTT258, this dynamic loop dually controlled its biological activities through distinct residues within the loop and this regulation provides a new model for the spatiotemporal control over FTT258 and potentially homologous APT function.     

Base cleavage specificity of angiogenin with Saccharomyces cerevisiae and Escherichia coli 5S RNAs

The base cleavage specificity of angiogenin toward naturally occurring polyribonucleotides has been determined by using rapid RNA sequencing technology. With 5S RNAs from Saccharomyces cerevisiae and Escherichia coli, angiogenin cleaves phosphodiester bonds exclusively at cytidylic or uridylic residues, preferably when the pyrimidines are followed by adenine. However, not all of the existent pyrimidine bonds in the 5S RNAs are cleaved, likely owing to elements of structure in the substrate. Despite the high degree of sequence homology between angiogenin and ribonuclease A (RNase A), which includes all three catalytic as well as substrate binding residues, the cleavage patterns with natural RNAs are unique to each enzyme. Angiogenin significantly hydrolyzes certain bonds that are not appreciably attacked by RNase A and vice versa. The different cleavage specificities of angiogenin and RNase A may account for the fact that the former is angiogenic while the latter is not.     

Angiogenin and its role in angiogenesis]

The review is devoted to angiogenin, one of the factors that induce formation of blood vessels, which is unique among them in that it is a ribonuclease. Consideration is given to the tertiary structure of human angiogenin; the catalytic and cell-receptor binding sites, their significance for angiogenic activity; the human angiogenin gene structure, chromosomal localization, and expression; the specificity of angiogenin as a ribonuclease and abolishment of protein synthesis; the nuclear localization of angiogenin in proliferating endothelial cells and its significance for angiogenic activity; angiogenin binding to a cell-surface actin as a plausible mechanism of inducing neovascularization (enhancement of plasminogen activation by actin with angiogenin, stimulation of the cell-associated proteolytic activity by angiogenin; promotion of the cultured cells invasiveness); modulation of mitogenic stimuli in endothelial, smooth muscle, and fibroblast cells by angiogenin. The importance of angiogenin as an adhesive molecule for endothelial and tumor cells is discussed too, as well as the modulation of tubular morphogenesis by bovine angiogenin, prevention of tumor growth in vivo by angiogenin antagonists, prospects of the use of angiogenin and angiogenin-encoding recombinant plasmids and vaccinia virus in therapeutic practice.     

Long-range interactions in the dimer interface of ornithine decarboxylase are important for enzyme function.

Ornithine decarboxylase (ODC) is a pyridoxal 5'-phosphate dependent enzyme that catalyzes the first committed step in the biosynthesis of polyamines. ODC is a proven drug target for the treatment of African sleeping sickness. The enzyme is an obligate homodimer, and the two identical active sites are formed at the dimer interface. Alanine scanning mutagenesis of dimer interface residues in Trypanosoma brucei ODC was undertaken to determine the energetic contribution of these residues to subunit association. Twenty-three mutant enzymes were analyzed by analytical ultracentrifugation, and none of the mutations were found to cause a greater than 1 kcal/mol decrease in dimer stability. These data suggest that the energetics of the interaction may be distributed across the interface. Most significantly, many of the mutations had large effects (DeltaDeltaG kcat/Km > 2.5 kcal/mol) on the catalytic efficiency of the enzyme. Residues that affected activity included those in or near the substrate binding site but also a number of residues that are distant (15-20 A) from this site. These data provide evidence that long-range energetic coupling of interface residues to the active site is essential for enzyme function, even though structural changes upon ligand binding to wild-type ODC are limited to local conformational changes in the active site. The ODC dimer interface appears to be optimized for catalytic function and not for dimer stability. Thus, small molecules directed to the ODC interfaces could impact biological function without having to overcome the difficult energetic barrier of dissociating the interacting partners.     

Crystal structure of the catalytic trimer of Methanococcus jannaschii aspartate transcarbamoylase

The catalytic trimer of Methanococcus jannaschii aspartate transcarbamoylase is extremely heat stable, maintaining 75% of its activity after heat treatment for 60 min at 75 degrees C. We undertook its structural analysis in order to understand the molecular basis of its thermostability and gain insight on how its catalytic function adapts to high temperature. Several structural elements potentially contributing to thermostability were identified. These include: (i) changes in the amino acid composition such as a decrease in the thermolabile residues Gln and Asn, an increase in the charged residues Lys and Glu, an increase in Tyr and a decrease in Ala residues; (ii) a larger number of salt bridges, in particular, the improvement of ion-pair networks; (iii) shortening of the N-terminus and shortening of three loops. An interesting feature of the crystal structure is the association of two crystallographically independent catalytic subunits into a staggered complex with an intertrimer distance of 33.8 A. The active site appears similar to Escherichia coli. Upon substrate binding, smaller changes in the global orientation of domains and larger conformational changes of the active site residues are expected as compared to E. coli.     

A hybrid of bovine pancreatic ribonuclease and human angiogenin: an external loop as a module controlling substrate specificity?

A comparison of the sequences of three homologous ribonucleases (RNase A, angiogenin and bovine seminal RNase) identifies three surface loops that are highly variable between the three proteins. Two hypotheses were contrasted: (i) that this variation might be responsible for the different catalytic activities of the three proteins; and (ii) that this variation is simply an example of surface loops undergoing rapid neutral divergence in sequence. Three hybrids of angiogenin and bovine pancreatic ribonuclease (RNase) A were prepared where regions in these loops taken from angiogenin were inserted into RNase A. Two of the three hybrids had unremarkable catalytic properties. However, the RNase A mutant containing residues 63-74 of angiogenin had greatly diminished catalytic activity against uridylyl-(3'----5')-adenosine (UpA), and slightly increased catalytic activity as an inhibitor of translation in vitro. Both catalytic behaviors are characteristic of angiogenin. This is one of the first examples of an engineered external loop in a protein. Further, these results are complementary to those recently obtained from the complementary experiment, where residues 59-70 of RNase were inserted into angiogenin [Harper and Vallee (1989) Biochemistry, 28, 1875-1884]. Thus, the external loop in residues 63-74 of RNase A appears to behave, at least in part, as an interchangeable 'module' that influences substrate specificity in an enzyme in a way that is isolated from the influences of other regions in the protein.     

Biochemical characterization of the chondroitinase B active site

Chondroitinase B from Flavobacterium heparinum is the only known lyase that cleaves the glycosaminoglycan, dermatan sulfate (DS), as its sole substrate. A recent co-crystal structure of chondroitinase B with a disaccharide product of DS depolymerization has provided some insight into the location of the active site and suggested potential roles of some active site residues in substrate binding and catalysis. However, this co-crystal structure was not representative of the actual enzyme-substrate complex, because the disaccharide product did not have the right length or the chemical structure of the minimal substrate (tetrasaccharide) involved in catalysis. Therefore, only a limited picture of the functional role of active site residues in DS depolymerization was presented in previous structural studies. In this study, by docking a DS tetrasaccharide into the proposed active site of the enzyme, we have identified novel roles of specific active site amino acids in the catalytic function of chondroitinase B. Our conformational analysis also revealed a unique, symmetrical arrangement of active site amino acids that may impinge on the catalytic mechanism of action of chondroitinase B. The catalytic residues Lys-250, Arg-271, His-272, and Glu-333 along with the substrate binding residues Arg-363 and Arg-364 were mutated using site-directed mutagenesis, and the kinetics and product profile of each mutant were compared with recombinant chondroitinase B. Mutating Lys-250 to alanine resulted in inactivation of the enzyme, potentially attributable to the role of the residue in stabilizing the carbanion intermediate formed during enzymatic catalysis. The His-272 and Glu-333 mutants showed diminished enzymatic activity that could be indicative of a possible role for one or both residues in the abstraction of the C-5 proton from the galactosamine. In addition, the Arg-364 mutant had an altered product profile after exhaustive digestion of DS, suggesting a role for this residue in defining the substrate specificity of chondroitinase B.     

Linkers of secondary structures in proteins.

Linkers that connect repeating secondary structures fall into conformational classes based on distance and main-chain torsion clustering. A data set of 300 unique protein chains with low pairwise sequence identity was clustered into only a few groups representing the preferred motifs. The linkers of two to eight residues for the nonredundant data set are designated H-Ln-H, H-Ln-E, E-Ln-H, E-Ln-E, where n is the length, H stands for alpha-helices, and E for beta-strands. Most of the clusters identified here corroborate earlier findings. However, 19 new clusters are identified in this paper, with many of them having seven and eight residue linkers. In our first analysis, the secondary structures flanking the linkers are both interacting and noninteracting and there is no precise angle of orientation between them. A second analysis was performed on a set of proteins with restricted orientations for the flanking elements, namely, mainly alpha class of proteins with orthogonal architecture. Two definite clusters are identified, one corresponding to linkers of orthogonal helices and the other to linkers of antiparallel helices. Loops forming binding sites or involved in catalytic activity are important determinants of the function of proteins. Although the structural conservation of the residues around the catalytic triad of serine proteases has been studied widely, there has not been a systematic analysis of the conformation of the loops that contain them. Residues of the catalytic triad reside in the linkers of beta-strands, with varying lengths of more than eight residues. Here, we analyze the structural conservation of such linkers by superposition, and observe a conserved structural feature of the linkers incorporating each of the three residues of the catalytic triad.     

High tolerance to mutations in a Chlamydia trachomatis peptide deformylase loop

AIM: To determine if and how a loop region in the peptide deformylase (PDF) of Chlamydia trachomatis regulates enzyme function.METHODS: Molecular dynamics simulation was used to study a structural model of the chlamydial PDF (cPDF) and predict the temperature factor per residue for the protein backbone atoms. Site-directed mutagenesis was performed to construct cPDF variants. Catalytic properties of the resulting variants were determined by an enzyme assay using formyl-Met-Ala-Ser as a substrate.RESULTS: In silico analysis predicted a significant increase in atomic motion in the DGELV sequence (residues 68-72) of a loop region in a cPDF mutant, which is resistant to PDF inhibitors due to two amino acid substitutions near the active site, as compared to wild-type cPDF. The D68R and D68R/E70R cPDF variants demonstrated significantly increased catalytic efficiency. The E70R mutant showed only slightly decreased efficiency. Although deletion of residues 68-72 resulted in a nearly threefold loss in substrate binding, this deficiency was compensated for by increased catalytic efficiency.CONCLUSION: Movement of the DGELV loop region is involved in a rate-limiting conformational change of the enzyme during catalysis. However, there is no stringent sequence requirement for this region for cPDF enzyme activity.     

Structural and Kinetic Analysis of Free Methionine-R-sulfoxide Reductase from Staphylococcus aureus: CONFORMATIONAL CHANGES DURING CATALYSIS AND IMPLICATIONS FOR THE CATALYTIC AND INHIBITORY MECHANISMS*

Free methionine-R-sulfoxide reductase (fRMsr) reduces free methionine R-sulfoxide back to methionine, but its catalytic mechanism is poorly understood. Here, we have determined the crystal structures of the reduced, substrate-bound, and oxidized forms of fRMsr from Staphylococcus aureus. Our structural and biochemical analyses suggest the catalytic mechanism of fRMsr in which Cys¹⁰² functions as the catalytic residue and Cys⁶⁸ as the resolving Cys that forms a disulfide bond with Cys¹⁰². Cys⁷⁸, previously thought to be a catalytic Cys, is a non-essential residue for catalytic function. Additionally, our structures provide insights into the enzyme-substrate interaction and the role of active site residues in substrate binding. Structural comparison reveals that conformational changes occur in the active site during catalysis, particularly in the loop of residues 97–106 containing the catalytic Cys¹⁰². We have also crystallized a complex between fRMsr and isopropyl alcohol, which acts as a competitive inhibitor for the enzyme. This isopropyl alcohol-bound structure helps us to understand the inhibitory mechanism of fRMsr. Our structural and enzymatic analyses suggest that a branched methyl group in alcohol seems important for competitive inhibition of the fRMsr due to its ability to bind to the active site.     

Biological and structural features of murine angiogenin-4, an angiogenic protein

Murine angiogenin-4 (mAng-4) is a member of the pancreatic ribonuclease superfamily that is expressed in some endodermally derived organs. We now show that mAng-4 is angiogenic using a thoracic aorta assay never before applied to the angiogenins. mAng-4, human angiogenin (hAng), and murine angiogenin-1 (mAng-1) stimulate the proliferation of IGR1 melanoma cells but do not stimulate the proliferation or migration of bovine corneal endothelial cells or primary mouse embryonic fibroblasts. In addition, we report the 3-D structure of mAng-4 at 2.02-A resolution. The structure shows that the residues forming the putative B1, P1, and B2 RNA-binding subsites occupy positions similar to their hAng counterparts. The B1 subsite is obstructed by Glu115 and Ile118. The obstruction is stabilized by a novel salt bridge between the C-terminal carboxyl group and the side chain of Arg99. Through mutational studies, we identify residues critical to the angiogenic function of mAng-4. The effect of H12A and H112A mutations in the catalytic site indicates that ribonucleolytic activity is essential to angiogenesis. The consequences of a nearby E115A mutation are consistent with a significant role for Glu115 in the attenuation of enzymatic activity but also suggest that sufficient suppression of catalysis is necessary for angiogenesis. The effect of an R32A mutation in the putative nuclear localization sequence indicates that this residue is crucial for angiogenesis. In the putative cell-binding segment, the replacement of Lys59 with Asn (its counterpart at position 61 of hAng) does not abrogate enzymatic activity but abolishes angiogenic activity, the reason for which is unclear.