Substrate-binding Region Research Articles

A Novel Point Mutation in P450c17 (CYP17) Causing Combined 17α-Hydroxylase/17,20-Lyase Deficiency

Combined 17alpha-hydroxylase/17,20-lyase deficiency is a rare cause of congenital adrenal hyperplasia and hypogonadism. Novel single amino acid changes in P450c17 provide potentially important insights into key structural domains for enzyme function. We report a novel missense mutation in P450c17 in a 17-yr-old female presenting with a malignant mixed germ cell tumor with yolk sac elements who demonstrated clinical and biochemical features of combined 17alpha-hydroxylase/17,20-lyase deficiency. Quantitative urinary steroid analysis was performed by high resolution gas chromatography. All eight coding exons of CYP17 were PCR amplified and sequenced. The position of arginine at codon 96 was modeled using the CYP17 structure 2c17 (www.rcsb.org). The CYP17 genes were subcloned into pcDNA3, expressed in HEK-293 cells, and chromatographed. 17alpha-Hydroxylase deficiency was confirmed by marked reductions in urinary and serum cortisol, androgens, and estradiol. Mutational analysis revealed a novel homozygous R96Q missense mutation in P450c17, affecting an amino acid in a key substrate-binding region of the enzyme, leading to complete inactivity. The description of a second missense mutation at codon 96 (R96W and R96Q) in the substrate-binding region of P450c17 provides strong evidence for the key role of this amino acid in 17alpha-hydroxylase/17,20-lyase function. An association between a malignant germ cell tumor and 17alpha-hydroxylase deficiency has not been reported previously, although the presence of gonadoblastoma in the ovary of a patient with this condition has recently been described.

Selectivity and potency of cyclin-dependent kinase inhibitors

Members of the cyclin-dependent kinase (CDK) family play key roles in various cellular processes. There are 11 members of the CDK family known till now. CDKs are activated by forming noncovalent complexes with cyclins such as A-, B-, C-, D- (D1, D2, and D3), and E-type cyclins. Each isozyme of this family is responsible for particular aspects (cell signaling, transcription, etc) of the cell cycle, and some of the CDK isozymes are specific to certain kinds of tissues. Aberrant expression and overexpression of these kinases are evidenced in many disease conditions. Inhibition of isozymes of CDKs specifically can yield beneficiary treatment modalities with minimum side effects. More than 80 3-dimensional structures of CDK2, CDK5, and CDK6 complexed with inhibitors have been published. This review provides an understanding of the structural aspects of CDK isozymes and binding modes of various known CDK inhibitors so that these kinases can be better targeted for drug discovery and design. The amino acid residues that constitute the cyclin binding region, the substrate binding region, and the area around the adenosine triphosphate (ATP) binding site have been compared for CDK isozymes. Those amino acids at the ATP binding site that could be used to improve the potency and subtype specificity have been described.

Design of asymmetric DNAzymes for dynamic control of nanoparticle aggregation states in response to chemical stimuli

Dynamic control of nanomaterial assembly states in response to chemical stimuli is critical in making multi-component materials with interesting properties. Previous work has shown that a Pb2+-specific DNAzyme allowed dynamic control of gold nanoparticle aggregation states in response to Pb2+, and the resulting color change from blue aggregates to red dispersed particles can be used as a convenient way of sensing Pb2+. However, a small piece of DNA (called invasive DNA) and low ionic strength (approximately 30 mM) were required for the process, limiting the scope of application in assembly and sensing. To overcome this limitation, a series of asymmetric DNAzymes, in which one of the two substrate binding regions is longer than the other, has been developed. With such a system, we demonstrated Pb2+-induced disassembly of gold nanoparticle aggregates and corresponding color change at room temperature without the need for invasive DNA, while also making the system more tolerant to ionic strength (33-100 mM). The optimal lengths of the long and short arms were determined to be 14 and 5 base pairs, respectively. In nanoparticle aggregates, the activity of the DNAzyme increased with decreasing ionic strength of the reaction buffer. This simpler and more versatile system allows even better dynamic control of nanoparticle aggregation states in response to chemical stimuli such as Pb2+, and can be used in a wider range of applications for colorimetric sensing of metal ions.

Structure of Chlorobium tepidum Sepiapterin Reductase Complex Reveals the Novel Substrate Binding Mode for Stereospecific Production of l-threo-Tetrahydrobiopterin

Sepiapterin reductase (SR) is involved in the last step of tetrahydrobiopterin (BH(4)) biosynthesis by reducing the di-keto group of 6-pyruvoyl tetrahydropterin. Chlorobium tepidum SR (cSR) generates a distinct BH(4) product, L-threo-BH(4) (6R-(1'S,2'S)-5,6,7,8-BH(4)), whereas animal enzymes produce L-erythro-BH(4) (6R-(1'R,2'S)-5,6,7,8-BH(4)) although it has high amino acid sequence similarities to the other animal enzymes. To elucidate the structural basis for the different reaction stereospecificities, we have determined the three-dimensional structures of cSR alone and complexed with NADP and sepiapterin at 2.1 and 1.7 A resolution, respectively. The overall folding of the cSR, the binding site for the cofactor NADP(H), and the positions of active site residues were quite similar to the mouse and the human SR. However, significant differences were found in the substrate binding region of the cSR. In comparison to the mouse SR complex, the sepiapterin in the cSR is rotated about 180 degrees around the active site and bound between two aromatic side chains of Trp-196 and Phe-99 so that its pterin ring is shifted to the opposite side, but its side chain position is not changed. The swiveled sepiapterin binding results in the conversion of the side chain configuration, exposing the opposite face for hydride transfer from NADPH. The different sepiapterin binding mode within the conserved catalytic architecture presents a novel strategy of switching the reaction stereospecificities in the same protein fold.

Structure of MurF from Streptococcus pneumoniae co‐crystallized with a small molecule inhibitor exhibits interdomain closure

In a broad genomics analysis to find novel protein targets for antibiotic discovery, MurF was identified as an essential gene product for Streptococcus pneumonia that catalyzes a critical reaction in the biosynthesis of the peptidoglycan in the formation of the cell wall. Lacking close relatives in mammalian biology, MurF presents attractive characteristics as a potential drug target. Initial screening of the Abbott small-molecule compound collection identified several compounds for further validation as pharmaceutical leads. Here we report the integrated efforts of NMR and X-ray crystallography, which reveal the multidomain structure of a MurF-inhibitor complex in a compact conformation that differs dramatically from related structures. The lead molecule is bound in the substrate-binding region and induces domain closure, suggestive of the domain arrangement for the as yet unobserved transition state conformation for MurF enzymes. The results form a basis for directed optimization of the compound lead by structure-based design to explore the suitability of MurF as a pharmaceutical target.

Glyoxalase I from Leishmania donovani: A potential target for anti-parasite drug

Glyoxalases are involved in a ubiquitous detoxification pathway. In pursuit of a better understanding of the biological function of the enzyme, the recombinant glyoxalase I (LdGLOI) protein has been characterized from Leishmania donovani, the most important pathogenic Leishmania species that is responsible for visceral leishmaniasis. A 24 kDa protein was heterologously expressed in Escherichia coli. LdGLOI showed a marked substrate specificity for trypanothione hemithioacetal over glutathione hemithioacetal. Antiserum against recombinant LdGLOI protein could detect a band of anticipated size ∼16 kDa in promastigote extracts. Several inhibitors of human GLOI showed that they are weak inhibitors of L. donovani growth. Overexpression of GLOI gene in L. donovani using Leishmania expression vector pspα hygroα, we detected elevated expression of GLOI RNA and protein. Comparative modelling of the 3-D structure of LDGLOI shows that substrate-binding region of the model involves important differences compared to the homologues, such as E. coli, specific to glutathione. Most notably a substrate-binding loop of LDGLOI is characterized by a deletion of five residues compared to the E. coli homologue. Further, a critical Arg in the E. coli variant at the substrate-binding site is replaced by Tyr in LDGLOI. These major differences result in entirely different shapes of the substrate-binding loop and presence of very different chemical groups in the substrate-binding site of LDGLOI compared to E. coli homologue suggesting an explanation for the difference in the substrate specificity. Difference in the substrate specificity of the human and LDGLOI enzyme could be exploited for structure-based drug designing of selective inhibitors against the parasite.

Crystal Structures of the Main Peptidase from the SARS Coronavirus Inhibited by a Substrate-like Aza-peptide Epoxide

The main peptidase (Mpro) from the coronavirus (CoV) causing severe acute respiratory syndrome (SARS) is one of the most attractive molecular targets for the development of anti-SARS agents. We report the irreversible inhibition of SARS-CoV Mpro by an aza-peptide epoxide (APE; kinact/Ki=1900(±400) M−1 s−1). The crystal structures of the Mpro:APE complex in the space groups C2 and P212121 revealed the formation of a covalent bond between the catalytic Cys145 Sγ atom of the peptidase and the epoxide C3 atom of the inhibitor, substantiating the mode of action of this class of cysteine-peptidase inhibitors. The aza-peptide component of APE binds in the substrate-binding regions of Mpro in a substrate-like manner, with excellent structural and chemical complementarity. In addition, the crystal structure of unbound Mpro in the space group C2 revealed that the “N-fingers” (N-terminal residues 1 to 7) of both protomers of Mpro are well defined and the substrate-binding regions of both protomers are in the catalytically competent conformation at the crystallization pH of 6.5, contrary to the previously determined crystal structures of unbound Mpro in the space group P21.

Calmodulin binding to the small GTPase Ral requires isoprenylated Ral

Ral, a member of the Ras-p21 superfamily of small GTPases, has been shown to require the calcium-signaling protein calmodulin (CaM) for activation. In the present work, we investigated the properties of the Ral-CaM interaction. Using CaM affinity binding assay with lysates from mammalian cells overexpressing various Ral mutants, we found that RalB(V23, ΔC AAX) lacking the C-terminal isoprenylation region bound significantly less efficiently to CaM. Binding of other mutants containing critical amino acid changes in the nucleotide or substrate binding regions (residues 23, 28, and 49) was not affected. In addition, all mutants bound significantly better in the presence of calcium versus the calcium chelator EGTA. Using in vitro transcription–translation in the presence of geranylgeranyl pyrophosphate, we demonstrate enhanced Ral binding to CaM. Inhibition of isoprenylation in cells in culture with lovastatin resulted in decreased binding of CaM to Ral. The present results show that post-translational isoprenylation of Ral is important in Ral–CaM interaction.

Crystal structure of the complex of group I PLA2 with a group II-specific peptide Leu-Ala-Ile-Tyr-Ser (LAIYS) at 2.6 Å resolution

Phospholipases A2s (PLA2s) are widely distributed in mammals and snake venoms. They catalyze the production of arachidonic acid from membrane phospholipids leading to the bioynthesis of pro-inflammatory eicosanoids. A peptide Leu-Ala-Ile-Tyr-Ser (LAIYS) was designed and synthesized as a specific inhibitor of PLA2. It was shown earlier that the peptide bound to group II PLA2 specifically and had a dissociation constant (Kd) of 8.8 × 10− 9 M. In the present studies for the binding of LAIYS with a group I PLA2 from Naja naja sagittifera using surface plasmon resonance the dissociation constant was found to be 4.5 × 10− 5 M which is considerably lower than the value found for the group II PLA2. In order to determine the details of binding at the molecular level, a group I PLA2 from the venom of Naja naja sagittifera was crystallized with peptide LAIYS. The crystal structure showed the presence of LAIYS at the substrate-binding site but has fewer interactions than those observed with group II PLA2 from Daboia russelli pulchella. The observed difference in the binding affinity is caused primarily due to poor fitting of the peptide LAIYS in the binding site of group I PLA2. Apparently, the location of Trp 19 in group I PLA2 is not favourable for the binding of LAIYS. The two complexes also differ drastically in the formation of intermolecular interactions. In the present structure, the side chain of Ser (P) interacts with His 48 and Asp 49 while in the complex with group II PLA2 it was Tyr (P) OH that formed the corresponding interactions. Tyr (P) in group I PLA2 is the main contributor of the hydrophobic interactions whereas in the complex of LAIYS with group II PLA2 it was the peptide segment Leu-Ala-Ile that produced the bulk of hydrophobic forces. The structures further showed that the peptide LAIYS was fully inside the substrate-binding region of the group II PLA2 while a significant portion of the peptide LAIYS was hanging outside the surface of the group I PLA2. The buried area in the complex with group II PLA2 was 811 Å2 whereas, the corresponding area in group I PLA2 was 449 Å2. This shows that the peptide LAIYS is very compatible with the substrate-binding site of group II PLA2 and rather poorly fits into the substrate-binding site of group I PLA2. This indicates that a highly specific ligand for one form of PLA2 may be a poor partner for another form of enzyme.

Structural Analysis of Escherichia Coli ThiF

Escherichia coli ThiF is an enzyme in the biosynthetic cascade for generating the essential cofactor thiamin pyrophosphate. In this cascade, ThiF catalyzes adenylation of the C terminus of ThiS. We report here the crystal structures of ThiF, alone and in complex with ATP. The structures provide insight into a preference for ATP during adenylation of the protein ThiS. Additionally, the structures reveal an ordered crossover loop predicted to clamp the flexible tail of ThiS into the ThiF active site during the adenylation reaction. The importance of the crossover loop for ThiF activity is highlighted by mutational analysis. Comparison of ThiF with the structural homologues MoeB, APPBP1-UBA3, and SAE1-SAE2 reveals that the ATP-binding site, including an arginine-finger, is maintained throughout evolution, and shows divergence occurring in protein substrate-binding sites and regions devoted to unique steps in the specific function of each enzyme.

Photoaffinity Labeling of P-Glycoprotein

The aim of the present review is to summarize recent progress in identifying substrate binding domains of P-glycoprotein by photoaffinity labeling. Preferred substrate binding regions have been identified using a number of photoaffinity ligands, including anthracyclines, the quinazoline iodoarylazidoprazosine (IAAP), dihydropyridines, taxanes and propafenones. These studies allowed identification of protein regions, which are involved in ligand interaction.

The molecular mechanisms underlying BiP-mediated gating of the Sec61 translocon of the endoplasmic reticulum

The Sec61 translocon of the endoplasmic reticulum membrane forms an aqueous pore that is gated by the lumenal Hsp70 chaperone BiP. We have explored the molecular mechanisms governing BiP-mediated gating activity, including the coupling between gating and the BiP ATPase cycle, and the involvement of the substrate-binding and J domain–binding regions of BiP. Translocon gating was assayed by measuring the collisional quenching of fluorescent probes incorporated into nascent chains of translocation intermediates engaged with microsomes containing various BiP mutants and BiP substrate. Our results indicate that BiP must assume the ADP-bound conformation to seal the translocon, and that the reopening of the pore requires an ATP binding–induced conformational change. Further, pore closure requires functional interactions between both the substrate-binding region and the J domain–binding region of BiP and membrane proteins. The mechanism by which BiP mediates translocon pore closure and opening is therefore similar to that in which Hsp70 chaperones associate with and dissociate from substrates.

Subtype-Specific Affinity for Corticosterone of Rat Organic Cation Transporters rOCT1 and rOCT2 Depends on Three Amino Acids within the Substrate Binding Region

The affinity of corticosterone to organic cation transporters (OCTs) is subtype- and species-dependent. For example, the IC50 values for corticosterone inhibition of cation uptake by transporters rOCT1 and rOCT2 are approximately 150 and approximately 4 microM, respectively. By introducing domains and amino acids from rOCT2 into rOCT1, we found that the exchange of three amino acids in the presumed 10th transmembrane alpha helix is sufficient to increase the affinity of rOCT1 for corticosterone to that of rOCT2. Replacement of these amino acids in rOCT2 decreased the affinity for corticosterone. These amino acids (Ala443, Leu447, and Gln448 in rOCT1 and Ile443, Tyr447, and Glu448 in rOCT2) are probably located within the substrate binding region because in rOCT1 mutants, the K(m) values for uptake of tetraethylammonium (TEA) and 1-methyl-4-phenylpyridinium (MPP) were decreased in parallel with a decrease of the IC50 values for the inhibition of cation uptake by corticosterone. In mutant rOCT1(L447Y/Q448E), the IC50 value for the inhibition of [3H]MPP (0.1 microM) uptake by corticosterone (24 +/- 4 microM) was significantly higher compared with the IC50 value for inhibition of [14C]TEA (10 microM) uptake (5.3 +/- 1.7 microM). This finding suggests an allosteric interaction between transported cation and corticosterone. Because this substrate-specific effect cannot be explained by differential replacement of corticosterone by MPP versus TEA and was observed after point mutations within the presumed substrate region, the data suggest that MPP or TEA bind to the substrate binding region simultaneously with corticosterone and cause a short-range allosteric effect on the corticosterone binding site.

Amino Acids Critical for Substrate Affinity of Rat Organic Cation Transporter 1 Line the Substrate Binding Region in a Model Derived from the Tertiary Structure of Lactose Permease

To identify functionally relevant amino acids in the rat organic cation transporter 1 (rOCT1), 18 consecutive amino acids in the presumed fourth transmembrane alpha helix (TMH) were mutated and functionally characterized after expression in Xenopus laevis oocytes. After mutation of three amino acids on successive turns of the alpha helix, K(m) values for tetraethylammonium (TEA) and/or 1-methyl-4-phenylpyridinium (MPP) were decreased. After replacement of Trp218 by tyrosine (W218Y) and Tyr222 by leucine (Y222L), the K(m) values for both TEA and MPP were decreased. In mutants Y222F and T226A, only the K(m) values for TEA and MPP were decreased, respectively. The data suggest that amino acids Trp218 and Tyr222 participate in the binding of both TEA and MPP, whereas Thr226 is only involved in the binding of MPP. Using the crystal structure of the lactose permease LacY from Escherichia coli that belongs to the same major facilitator superfamily as rOCT1, we modeled the tertiary structure of the presumed 12 transmembrane alpha helices. The validity of the model was suggested because seven amino acids that have been shown to participate in the binding of cations by mutagenesis experiments [fourth TMH Trp218, Tyr222, and Thr226 (this work); 10th TMH Ala443, Leu447, and Gln448 (companion work in this issue of Molecular Pharmacology); 11th TMH Asp475 (previous report)] are located in one region surrounding a large cleft that opens to the intracellular side. The dimensions of TEA in comparison with the interacting amino acids in the modeled cleft suggest that more than one TEA molecule can bind in parallel to the modeled conformation of the transporter.

Molecular Determinants of Substrate Recognition in Hematopoietic Protein-tyrosine Phosphatase

The extracellular signal-regulated protein kinase 2 (ERK2) plays a central role in cellular proliferation and differentiation. Full activation of ERK2 requires dual phosphorylation of Thr183 and Tyr185 in the activation loop. Tyr185 dephosphorylation by the hematopoietic protein-tyrosine phosphatase (HePTP) represents an important mechanism for down-regulating ERK2 activity. The bisphosphorylated ERK2 is a highly efficient substrate for HePTP with a kcat/Km of 2.6 x 10(6) m(-1) s(-1). In contrast, the kcat/Km values for the HePTP-catalyzed hydrolysis of Tyr(P) peptides are 3 orders of magnitude lower. To gain insight into the molecular basis for HePTP substrate specificity, we analyzed the effects of altering structural features unique to HePTP on the HePTP-catalyzed hydrolysis of p-nitrophenyl phosphate, Tyr(P) peptides, and its physiological substrate ERK2. Our results suggest that substrate specificity is conferred upon HePTP by both negative and positive selections. To avoid nonspecific tyrosine dephosphorylation, HePTP employs Thr106 in the substrate recognition loop as a key negative determinant to restrain its protein-tyrosine phosphatase activity. The extremely high efficiency and fidelity of ERK2 dephosphorylation by HePTP is achieved by a bipartite protein-protein interaction mechanism, in which docking interactions between the kinase interaction motif in HePTP and the common docking site in ERK2 promote the HePTP-catalyzed ERK2 dephosphorylation (approximately 20-fold increase in kcat/Km) by increasing the local substrate concentration, and second site interactions between the HePTP catalytic site and the ERK2 substrate-binding region enhance catalysis (approximately 20-fold increase in kcat/Km) by organizing the catalytic residues with respect to Tyr(P)185 for optimal phosphoryl transfer.

Structures of wild-type and P28L/Y173F tryptophan synthase α-subunits from Escherichia coli

The α-subunit of tryptophan synthase (αTS) catalyzes the cleavage of indole-3-glycerol phosphate to glyceraldehyde-3-phosphate and indole, which is used to yield the amino acid tryptophan in tryptophan biosynthesis. Here, we report the first crystal structures of wild-type and double-mutant P28L/Y173F α-subunit of tryptophan synthase from Escherichia coli at 2.8 and 1.8 Å resolution, respectively. The structure of wild-type αTS from E. coli was similar to that of the α 2β 2 complex structure from Salmonella typhimurium. As compared with both structures, the conformational changes are mostly in the interface of α- and β-subunits, and the substrate binding region. Two sulfate ions and two glycerol molecules per asymmetric unit bind with the residues in the active sites of the wild-type structure. Contrarily, double-mutant P28L/Y173F structure is highly closed at the window for the substrate binding by the conformational changes. The P28L substitution induces the exposure of hydrophobic amino acids and decreases the secondary structure that causes the aggregation. The Y173F suppresses to transfer a signal from the α-subunit core to the α-subunit surface involved in interactions with the β-subunit and increases structural stability.

Topology of the substrate-binding site of a Lys49-phospholipase A2 influences Ca2+-independent membrane-damaging activity.

BthTx-I (bothropstoxin-I) is a myotoxic Lys49-PLA2 (phospholipase A2 with Lys49) isolated from Bothrops jararacussu venom, which damages liposome membranes by a Ca2+-independent mechanism. The highly conserved Phe5/Ala102/Phe106 motif in the hydrophobic substrate-binding site of the Asp49-PLA2s is substituted by Leu5/Val102/Leu106 in the Lys49-PLA2s. The Leu5/Val102/Leu106 triad in BthTx-I was sequentially mutated via all single- and double-mutant combinations to the Phe5/Ala102/Phe106 mutant. All mutants were expressed as inclusion bodies in Escherichia coli, and the thermal stability (Tm), together with the myotoxic and Ca2+-independent membrane-damaging activities of the recombinant proteins, were evaluated. The far-UV CD profiles of the native, wild-type recombinant and the L106F (Leu106-->Phe) and L5F/F102A/L106F mutant proteins were identical. The L5F, V102A, L5F/V102A and V102A/L106F mutants showed distorted far-UV CD profiles; however, only the L5F and L5F/V102A mutants showed significant decreases in Tm. Alterations in the far-UV CD spectra correlated with decreased myotoxicity and protein-induced release of a liposome-entrapped marker. However, the V102A/L106F and L5F/V102A/L106F mutants, which presented high myotoxic activities, showed significantly reduced membrane-damaging activity. This demonstrates that the topology of the substrate-binding region of BthTx-I has a direct effect on the Ca2+-independent membrane damage, and implies that substrate binding retains an important role in this process.

Purification, identification and characterisation of seprase from bovine serum

The study and identification for the first time of a soluble form of a seprase activity from bovine serum is presented. To date, this activity has only been reported to be an integral membrane protease but has been known to shed from its membrane. The activity was purified 30,197-fold to homogeneity, using a combination of column chromatographies, from bovine serum. Inhibition by DFP, resulting in an IC 50 of 100:nM confirms classification as a serine protease. The protease after separation and visualisation by native PAGE was subjected to tryptic digestion and the subsequent peptides sequenced. Each peptide sequenced was found to be present in the primary structure of seprase/fibroblast activation protein (FAP), a serine gelatinase specific for proline-containing peptides and macromolecules. Substrate specificity studies using kinetic, RP-HPLC and LC–MS analysis of synthetic peptides suggest that this peptidase has an extended substrate-binding region in addition to the primary specificity site S 1. This analysis revealed at least five subsites to be involved in enzyme–substrate binding, with the smallest peptide cleaved being a tetrapeptide. A proline residue in position P 1 was absolutely necessary therefore showing high primary substrate specificity for the Pro-X bond, while a preference for a hydrophobic residue at the C-terminal end of the scissile bond (P 1′) was evident. The enzyme also showed complete insensitivity to the prolyl oligopeptidase specific inhibitors, JTP-4819, Fmoc-Ala-pyrrCN and Z-Phe-Pro-BT. To date, no physiological substrate has clearly been defined for this protease but its ability to effectively degrade gelatin suggests a candidate protein substrate in vivo and a possible role in extracellular matrix protein degradation.

Multiple Time Scale Backbone Dynamics of Homologous Thermophilic and Mesophilic Ribonuclease HI Enzymes*1

Backbone conformational fluctuations on multiple time scales in a cysteine-free Thermus thermophilus ribonuclease HI mutant (ttRNH∗) are quantified using 15N nuclear magnetic spin relaxation. Laboratory-frame relaxation data acquired at 310 K and at static magnetic field strengths of 11.7, 14.1 and 18.8 T are analysed using reduced spectral density mapping and model-free approaches. Chemical exchange line broadening is characterized using Hahn-echo transverse and multiple quantum relaxation data acquired over a temperature range of 290–320 K and at a static magnetic field strength of 14.1 T. Results for ttRNH∗ are compared to previously published data for a mesophilic homologue, Escherichia coli ribonuclease HI (ecRNH). Intramolecular conformational fluctuations on the picosecond-to-nanosecond time scale generally are similar for ttRNH∗ and ecRNH. β-Strands 3 and 5 and the glycine-rich region are more rigid while the substrate-binding handle region and C-terminal tail are more flexible in ttRNH∗ than in ecRNH. Rigidity in the two β-strands and the glycine-rich region, located along the periphery of the central β-sheet, may be associated with the increased thermodynamic stability of the thermophilic enzyme. Chemical exchange line broadening, reflecting microsecond-to-millisecond time scale conformational changes, is more pronounced in ttRNH∗ than in ecRNH, particularly for residues in the handle and surrounding the catalytic site. The temperature dependence of chemical exchange show an increase of ∼15 kJ/mol in the apparent activation energies for ttRNH∗ residues in the handle compared to ecRNH. Increased activation barriers, coupled with motion between α-helices B and C not present in ecRNH, may be associated with the reduced catalytic activity of the thermophilic enzyme at 310 K.

Multiple Time Scale Backbone Dynamics of Homologous Thermophilic and Mesophilic Ribonuclease HI Enzymes

Backbone conformational fluctuations on multiple time scales in a cysteine-free Thermus thermophilus ribonuclease HI mutant (ttRNH∗) are quantified using 15N nuclear magnetic spin relaxation. Laboratory-frame relaxation data acquired at 310 K and at static magnetic field strengths of 11.7, 14.1 and 18.8T are analysed using reduced spectral density mapping and model-free approaches. Chemical exchange line broadening is characterized using Hahn-echo transverse and multiple quantum relaxation data acquired over a temperature range of 290–320 K and at a static magnetic field strength of 14.1T. Results for ttRNH∗ are compared to previously published data for a mesophilic homologue, Escherichia coli ribonuclease HI (ecRNH). Intramolecular conformational fluctuations on the picosecond-to-nanosecond time scale generally are similar for ttRNH∗ and ecRNH. β-Strands 3 and 5 and the glycine-rich region are more rigid while the substrate-binding handle region and C-terminal tail are more flexible in ttRNH∗ than in ecRNH. Rigidity in the two β-strands and the glycine-rich region, located along the periphery of the central β-sheet, may be associated with the increased thermodynamic stability of the thermophilic enzyme. Chemical exchange line broadening, reflecting microsecond-to-millisecond time scale conformational changes, is more pronounced in ttRNH∗ than in ecRNH, particularly for residues in the handle and surrounding the catalytic site. The temperature dependence of chemical exchange show an increase of ∼15 kJ/mol in the apparent activation energies for ttRNH∗ residues in the handle compared to ecRNH. Increased activation barriers, coupled with motion between α-helices B and C not present in ecRNH, may be associated with the reduced catalytic activity of the thermophilic enzyme at 310 K.