Blood clotting factor IX Niigata: substitution of alanine-390 by valine in the catalytic domain

Factor IX Niigata is a mutant factor IX responsible for the moderately severe hemophilia B in a patient who has a normal level of factor IX antigen with reduced clotting activity (1-4% of normal). We reported previously that the purified mutant protein could be converted to the factor IXa beta form by factor XIa/Ca2+ at a rate similar to that in the case of normal factor IX, but the resulting mutant factor IXa beta could not activate factor X in the presence of factor VIII, Ca2+, and phospholipids (Yoshioka, A. et al. (1986) Thromb. Res. 42, 595-604). In the present study, we analyzed factor IX Niigata at the structural level to elucidate the molecular abnormality responsible for the loss of clotting activity. Amino acid sequence analysis of a peptide obtained on lysyl endopeptidase digestion, coupled with subsequent SP-V8 digestion, demonstrated that the alanine at position 390 was substituted by valine in the catalytic domain of the factor IX Niigata molecule.     

Molecular basis of mucopolysaccharidosis type VII: replacement of Ala619 in beta-glucuronidase with Val

We have identified a mutation causing beta-glucuronidase (beta Gl) deficiency in a 6-year-old girl with mucopolysaccharidosis type VII. Enzyme assay of lysates of a girl's lymphocytes or cultured fibroblasts showed little residual activity and a normal beta Gl-specific mRNA level, as revealed by Northern-blot analysis. Sequencing of the full-length mutated cDNA revealed a C----T transition, an event causing a single Ala619----Val change (we designated this variant beta GGifu). This change is detected by loss of the cleavage site for the enzyme Fnu4HI in the mutated cDNA. On the basis of the loss of Fnu4HI restriction site, the patient was shown to be a homozygote with the beta GGifu mutation and her parents and brother were heterozygotes. This mutation disrupts a functional domain consisting of a region of sequence highly conserved among human, rat and bacterial beta Gls, and it reduces the enzyme activity, as tested by transfection of COS cells with expression vectors harboring the mutated cDNA.     

Substitution of Val20 by Gly in elongation factor Tu. Effects on the interaction with elongation factors Ts, aminoacyl-tRNA and ribosomes

Substitution of V20 by G in the consensus element G18HVDHGK24 of EF-Tu (referred to as EF-TuG20) strongly influences the interaction with GDP as well as the GTPase activity [Jacquet, E. & Parmeggiani, A. (1988) EMBO J. 7, 2861-2867]. In an extension of this work we describe additional properties of the mutated factor, paying particular attention to the interaction with the macromolecular ligands. Our results show that the conformational transitions induced by the mutation strongly favor the regeneration of the active complex EF-TuG20.GTP, almost as effectively as with wild-type EF-Tu in the presence of elongation factor Ts. Addition of elongation factor Ts further enhances the rate of the GDP to GTP exchange of the mutated factor. Remarkably, EF-TuG20.GDP can support the enzymatic binding of aminoacyl-tRNA to ribosome.mRNA at low MgCl2 concentration, an effect that with wild-type EF-Tu can only occur in the presence of kirromycin. Our results show that EF-TuG20.GDP shares common features with the GTP-like conformation induced by kirromycin on wild-type EF-Tu. The ability of the ribosome to activate the EF-TuG20 center for GTP hydrolysis is strongly decreased, while the stimulation by aminoacyl-tRNA is conserved. The ribosomal activity is partially restored by addition of aminoacyl-tRNA plus poly(U), showing that codon/anticodon interaction contribute to correct the anomalous interaction between ternary complex and ribosomes. The impaired activity of EF-TuG20 in poly(Phe) synthesis is related to the degree of defective GTP hydrolysis and, most interestingly, it is characterized by a striking increase of the fidelity of translation at high MgCl2 concentration. This effect probably depends on a more selective recognition of the ternary complex by ribosome.mRNA, as a consequence of a longer pausing of EF-TuG20 on the ribosome. In conclusion, position 20 in EF-Tu is important for coordinating the allosteric mechanisms controlling the action of EF-Tu and its ligands.     

Molecular defect of prothrombin Barcelona. Substitution of cysteine for arginine at residue 273

Prothrombin Barcelona has been isolated from a patient with a normal prothrombin antigen level but low prothrombin coagulant activity. The activation of this protein is impaired by the absence of one of the two factor Xa-catalyzed cleavages that normally lead to the formation of thrombin. Prothrombin Barcelona and prothrombin were isolated from patient plasma and normal plasma, respectively, in a single-step, high-yield immunoaffinity purification using conformation-specific antibodies immobilized on Sepharose. After reduction and alkylation, the purified proteins were subjected to trypsin hydrolysis. The resulting peptides were separated by reverse-phase high performance liquid chromatography. Comparison of the peptide maps of prothrombin Barcelona and prothrombin demonstrated that a peptide, identified as fragment 274-287 in prothrombin by automated Edman degradation, was missing in the prothrombin Barcelona digest. In the chromatogram derived from prothrombin Barcelona, an additional peptide was observed. The amino acid sequence of this peptide was Ala-Ile-Glu-Gly-Cys-Thr-Ala-Thr-Ser-Glu-Tyr-Gln-Thr-Phe-Phe-Asn-Pro-Arg, corresponding to residues 269-287 in prothrombin except for the substitution of cysteine for arginine at residue 273. The substitution of cysteine for arginine was confirmed by tryptic digestion of 14C-carboxymethylated prothrombin Barcelona. Edman degradation of fragment 269-287 indicated the association of 14C with the cysteine at residue 273. The replacement of arginine by cysteine at residue 273, adjacent to the known factor Xa cleavage site, precludes normal activation of prothrombin Barcelona by factor Xa and the generation of thrombin.     

Localization of a heparin binding site in the catalytic domain of factor XIa.

Inhibition of factor XIa by protease nexin II (K(i) approximately 450 pM) is potentiated by heparin (K(I) approximately 30 pM). The inhibition of the isolated catalytic domain of factor XIa demonstrates a similar potentiation by heparin (K(i) decreasing from 436 +/- 62 to 88 +/- 10 pM) and also binds to heparin on surface plasmon resonance (K(d) 11.2 +/- 3.2 nM vs K(d) 8.63 +/- 1.06 nM for factor XIa). The factor XIa catalytic domain contains a cysteine-constrained alpha-helix-containing loop: (527)CQKRYRGHKITHKMIC(542), identified as a heparin-binding region in other coagulation proteins. Heparin-binding studies of coagulation proteases allowed a grouping of these proteins into three categories: group A (binding within a cysteine-constrained loop or a C-terminal heparin-binding region), factors XIa, IXa, Xa, and thrombin; group B (binding by a different mechanism), factor XIIa and activated protein C; and group C (no binding), factor VIIa and kallikrein. Synthesized peptides representative of the factor XIa catalytic domain loop were used as competitors in factor XIa binding and inhibition studies. A native sequence peptide binds to heparin with a K(d) = 86 +/- 15 nM and competes with factor XIa in binding to heparin, K(i) = 241 +/- 37 nM. A peptide with alanine substitutions at (534)H, (535)K, (538)H, and (539)K binds and competes with factor XIa for heparin-binding in a manner nearly identical to that of the native peptide, whereas a scrambled peptide is approximately 10-fold less effective, and alanine substitutions at residues (529)K, (530)R, and (532)R result in loss of virtually all activity. We conclude that residues (529)K, (530)R, and (532)R comprise a high-affinity heparin-binding site in the factor XIa catalytic domain.     

Four loops of the catalytic domain of factor viia mediate the effect of the first EGF-like domain substitution on factor viia catalytic activity

The presence of tissue factor is essential for factor VIIa (FVIIa) to reach its full catalytic potential. The previous work in this laboratory demonstrated that substitution of the EGF1 domain of factor VIIa with that of factor IX (FVII((IXegf1))a) results in a substantial decrease in TF-binding affinity and catalytic activity. Supporting simulations of the solution structures of Ca(2+)-bound factor VIIa and FVII((IXegf1))a with tissue factor are provided. Mutants are generated, based on the simulation model, to study the effect of EGF1 substitution on catalytic activity. The simulations show larger Gla-EGF1 and EGF1-EGF2 inter-domain motions for FVII((IXegf1))a than for factor VIIa. The catalytic domain of the chimeric factor VIIa has been disturbed and several surface loops in the catalytic domain of FVII((IXegf1))a (Loop 170s (170-182), Loop 1 (185-188) and Loop 2 (221A-225)) manifest larger position fluctuations than wild-type. The position of Loop 140s (142-152) of FVII((IXegf1))a, near the N terminus insertion site of the catalytic domain, shifts relative to factor VIIa, resulting in a slight alteration of the active site. The results suggest that these four loops mediate the effect of the EGF1 domain substitution on the S1 site and catalytic residues. To test the model, we prepared mutations of these surface loops, including four FVII mutants, D186A, K188A, L144A and R147A, a FVII mutant with multiple mutations (MM3: L144A+R147A+D186A) and a FVII mutant with Loop 170s partially deleted, Loop 170s(del). The catalytic activities towards a small peptidyl substrate decreased 2.4, 4.5 and 9-fold for Loop 170s(del)a (a, activated), L144Aa and D186Aa, respectively, while MM3a lost almost all catalytic activity. The combined results of the simulations and mutants provide insight into the mechanism by which tissue factor enhances factor VIIa catalytic activity.     

Formation of factor 390 by cell extracts of Methanosarcina barkeri.

Cell extracts of Methanosarcina barkeri converted coenzyme F420 in an ATP-dependent reaction to the adenylylated derivative factor 390. Although it was reported previously (L. M. Gloss and R. P. Hausinger, BioFactors 1:237-240, 1988) that whole cells were unable to perform this conversion, we observed the conversion in 7 of 11 extracts, all of which were prepared from different batches of cells.     

Solution structure of the Cys74 to Ala74 mutant of the recombinant catalytic domain of Zoocin A

Zoocin A is a Zn‐metallopeptidase secreted by Streptococcus zooepidemicus strain 4881. Its catalytic domain is responsible for cleaving the D‐alanyl‐L‐alanine peptide bond in streptococcal peptidoglycan. The solution NMR structure of the Cys74 to Ala74 mutant of the recombinant catalytic domain (rCAT C74A) has been determined. With a previous structure determination for the recombinant target recognition domain (rTRD), this completes the 3D structure of zoocin A. While the structure of rCAT C74A resembles those of the catalytic domains of lysostaphin and LytM, the substrate binding groove is wider and no tyrosine residue was observed in the active site. Proteins 2016; 85:177–181. © 2016 Wiley Periodicals, Inc.     

Substitution of proline 82 by threonine induces autophosphorylating activity in GTP-binding domain of elongation factor Tu

Mutation of Pro82 into Thr, a residue situated in the second element (D80CPG83) of the consensus sequence proposed to interact with GTP/GDP in GTP-binding proteins was introduced via site-directed mutagenesis in the isolated guanine nucleotide-binding domain (G domain) of elongation factor Tu. G domainPT82 displays virtually no GTPase activity. As a major change, the apparent inhibition of the GTPase reaction is associated with the appearance of autophosphorylating activity, as in ras product p21 in the case of mutation Ala59----Thr, corresponding to 82 in elongation factor Tu. Dependence of this reaction on mono- and divalent cation concentration and on pH is essentially the same as for the GTPase of wild-type G domain. The autokinase reaction follows an apparent first order rate, suggesting an intermolecular mechanism. Analysis of amino acid and peptide composition of the 32P-labeled G domainPT82, as well as Edman degradation of the tryptic peptide containing the covalently bound 32P, shows that Thr82 is the phosphorylated residue. Taken together, these results point out that Thr82 is in close proximity to the gamma-phosphate of GTP, as in the case of Thr59 in p21. These results are in agreement with the observations derived from x-ray diffraction analysis that the tertiary structure of the GTP-binding domain of elongation factor Tu and that of p21 are similar.     

Insights on the structural perturbations in human MTHFR Ala222Val mutant by protein modeling and molecular dynamics

Methylenetetrahydrofolate reductase (MTHFR) protein catalyzes the only biochemical reaction which produces methyltetrahydrofolate, the active form of folic acid essential for several molecular functions. The Ala222Val polymorphism of human MTHFR encodes a thermolabile protein associated with increased risk of neural tube defects and cardiovascular disease. Experimental studies have shown that the mutation does not affect the kinetic properties of MTHFR, but inactivates the protein by increasing flavin adenine dinucleotide (FAD) loss. The lack of completely solved crystal structure of MTHFR is an impediment in understanding the structural perturbations caused by the Ala222Val mutation; computational modeling provides a suitable alternative. The three-dimensional structure of human MTHFR protein was obtained through homology modeling, by taking the MTHFR structures from Escherichia coli and Thermus thermophilus as templates. Subsequently, the modeled structure was docked with FAD using Glide, which revealed a very good binding affinity, authenticated by a Glide XP score of ?10.3983 (kcal mol^?1). The MTHFR was mutated by changing Alanine 222 to Valine. The wild-type MTHFR-FAD complex and the Ala222Val mutant MTHFR-FAD complex were subjected to molecular dynamics simulation over 50 ns period. The average difference in backbone root mean square deviation (RMSD) between wild and mutant variant was found to be ~.11 Å. The greater degree of fluctuations in the mutant protein translates to increased conformational stability as a result of mutation. The FAD-binding ability of the mutant MTHFR was also found to be significantly lowered as a result of decreased protein grip caused by increased conformational flexibility. The study provides insights into the Ala222Val mutation of human MTHFR that induces major conformational changes in the tertiary structure, causing a significant reduction in the FAD-binding affinity.     

Subdomain organization and catalytic residues of the F factor TraI relaxase domain

TraI from conjugative plasmid F factor is both a "relaxase" that sequence-specifically binds and cleaves single-stranded DNA (ssDNA) and a helicase that unwinds the plasmid during transfer. Using limited proteolysis of a TraI fragment, we generated a 36-kDa fragment (TraI36) retaining TraI ssDNA binding specificity and relaxase activity but lacking the ssDNA-dependent ATPase activity of the helicase. Further proteolytic digestion of TraI36 generates stable N-terminal 26-kDa (TraI26) and C-terminal 7-kDa fragments. Both TraI36 and TraI26 are stably folded and unfold in a highly cooperative manner, but TraI26 lacks affinity for ssDNA. Mutational analysis of TraI36 indicates that N-terminal residues Tyr(16) and Tyr(17) are required for efficient ssDNA cleavage but not for high-affinity ssDNA binding. Although the TraI36 N-terminus provides the relaxase catalytic residues, both N- and C-terminal structural domains participate in binding, suggesting that both domains combine to form the TraI relaxase active site.     

纤维素酶家族及其催化结构域分子改造的新进展

纤维素酶的分子改造是其催化性能改进及催化效率提升的重要手段。近年来,组学技术与结构测定技术的迅速发展,人们已建立了包括糖苷水解酶(Glycoside hydrolase,GH)在内的碳水化合物活性酶组分数据库。通过对同一蛋白家族进行序列比对、分子进化分析与祖先基因重构,以结构模建分析为指导的纤维素酶分子改造,可以明显缩小序列或结构的搜索空间,加快酶分子改造的速度,增大理性设计成功的概率;同时针对催化中心活性架构的分析可以进一步阐明纤维素酶的催化机理与酶分子持续性降解机制。文中主要对纤维素酶家族及其催化结构域的分子改造取得的最新进展作了综述。在后基因组时代基于蛋白质家族中的海量数据分析,以其保守结构信息为指导的理性设计,将会成为纤维素酶分子改造的重要方向,从而推动生物质转化工艺的快速发展。     

A Structured-based Model for the Decreased Activity of Ala222Val and Glu429Ala Methylenetetrahydrofolate Reductase (MTHFR) Mutants

The structure of human Methylenetetrahydrofolate Reductase (MTHFR) is not known either by NMR or by X-ray methods. Phosphorylation seems to play an important role in the functioning of this flavoprotein. MTHFR catalyzes an irreversible reaction in homocysteine metabolism. Phosphorylation decreases the activity of MTHFR by enhancing the sensitivity of the enzyme to SAdenosylmethione. Two common polymorphisms in MTHFR, Ala222Val and Glu429Ala, can result in a number of vascular diseases. Effects of the Glu429Ala polymorphism on the structure of human MTHFR remain undetermined due to limited structural information. Hence, structural models of the MTHFR mutants were constructed using I-TASSER and assessed by PROCHECK, DFIRE and Verify3D tools. A mechanism is further suggested for the decreased activity of the Ala222Val and Glu429Ala mutants due to a decrease in number of serine phosphorylation sites using information gleaned from the molecular models. This provides insights for the understanding of structure-function relationship for MTHFR.     

Deletion of the regulatory domain in the pyridoxal phosphate-dependent heme protein cystathionine beta-synthase alleviates the defect observed in a catalytic site mutant.

The most common cause of severely elevated homocysteine or homocystinuria is inherited disorders in cystathionine beta-synthase. The latter enzyme is a unique hemeprotein that catalyzes pyridoxal phosphate (PLP)-dependent condensation of serine and homocysteine to give cystathionine, thus committing homocysteine to catabolism. A point mutation, V168M, has been described in a homocystinuric cell line and is associated with a B(6)-responsive phenotype. In this study, we have examined the kinetic properties of this mutant and demonstrate that the mutation affects the PLP but not the heme content. The approximately 13-fold diminution in activity because of the mutation corresponds to an approximately 7-fold decrease in the level of bound PLP. This may be explained by half of the sites activity associated with cystathionine beta-synthase. The addition of PLP results in partial but not full restoration of activity to wild type levels. Elimination of the C-terminal quarter of the mutant protein results in alleviation of the catalytic penalty imposed by the V168M mutation. The resulting truncated protein is very similar to the corresponding truncated enzyme with wild type sequence and is now able to bind the full complement of both heme and PLP cofactors. These results indicate that the V168M mutation per se does not affect binding of PLP directly and that interactions between the regulatory C terminus and the catalytic N terminus are important in modulating the cofactor content and therefore the activity of the full-length enzyme. These studies provide the first biochemical explanation for the B(6)-responsive phenotype associated with a cystathionine beta-synthase-impaired homocystinuric genotype.     

Functional and structural effects of an Ala to Val mutation in the adenovirus serotype 2 fibre

H2ts125 is a fibre-defective, temperature-sensitive mutant of adenovirus serotype 2. H2ts125 fibre is unstable at the non-permissive temperature (ts phenotype), and does not migrate in the same way as the wild-type fibre in an SDS/polyacrylamide gel (elm phenotype). Sequence analysis has shown that H2ts125 carries two mutations on the fibre gene: Leu105 to Phe, and Ala434 to Val. Analysis of the structural modifications occurring in H2ts125 fibre was performed using peptide finger-printing and antipeptide sera as immunological probes. We found that all the detectable structural alterations in the mutant fibre were due to the substitution on codon 434. In addition, the ts phenotype was rescued by a wild-type DNA fragment containing the 3' moiety of the fibre gene and overlapping the 434th codon. Morphological analysis of fibre molecules observed under the electron microscope showed minor but statistically significant differences in the fibre length between mutant and wild-type. The mutant fibre was found to be slightly longer (308.8 +/- 1.9 A) than the wild-type fibre (300.1 +/- 2.1 A). Thus both ts and elm phenotypes were carried by the same Ala434 to Val mutation which probably resulted from a change in the three-dimensional structure of the fibre protein, and not from some proteolytic cleavage.     

Substitution Processes in Molecular Evolution. III. Deleterious Alleles

The substitution processes for various models of deleterious alleles are examined using computer simulations and mathematical analyses. Most of the work focuses on the house-of-cards model, which is a popular model of deleterious allele evolution. The rate of substitution is shown to be a concave function of the strength of selection as measured by α = 2Nσ, where N is the population size and σ is the standard deviation of fitness. For α<1, the house-of-cards model is essentially a neutral model; for α>4, the model ceases to evolve. The stagnation for large α may be understood by appealing to the theory of records. The house-of-cards model evolves to a state where the vast majority of all mutations are deleterious, but precisely one-half of those mutations that fix are deleterious (the other half are advantageous). Thus, the model is not a model of exclusively deleterious evolution as is frequently claimed. It is argued that there are no biologically reasonable models of molecular evolution where the vast majority of all substitutions are deleterious. Other models examined include the exponential and gamma shift models, the Hartl-Dykhuizen-Dean (HDD) model, and the optimum model. Of all those examined, only the optimum and HDD models appear to be reasonable candidates for silent evolution. None of the models are viewed as good candidates for protein evolution, as none are both biologically reasonable and exhibit the variability in substitutions commonly observed in protein sequence data.     

Apolactoferrin inhibits the catalytic domain of matrix metalloproteinase-2 by zinc chelation.

Lactoferrin (LTF) is a multifunctional iron-binding protein that is also capable of binding other divalent metal cations, especially Zn2+. Recent investigations indicate that lactoferrin levels are elevated in many disease conditions in which matrix metalloproteinases (MMPs), particularly MMP-2, are also elevated, suggesting that the 2 proteins may interact. This possibility was examined by determining the effect of LTF in its holo (metal-bound) and apo (metal-free) forms on the proteolytic activity of MMP-2 and other similar zinc metalloproteases. Pre-incubation with apolactoferrin, but not hololactoferrin, greatly reduced the hydrolysis of a peptide substrate by MMP-2, but not by MMP-1, -8, -9, or -13. This inhibition was specific for the 42 kDa catalytic domain fragment of MMP-2 lacking the hemopexin domain, since the 66 kDa form was poorly inhibited by apolactoferrin. The inhibition of the MMP-2 catalytic domain was strongly temperature sensitive, indicating that the conformation of one or both proteins is crucial to this interaction. To ascertain the mechanism of inhibition, increasing concentrations of ZnCl2 and FeCl2 were added to the reaction. While addition of Fe2+ did not reverse inhibition, the addition of Zn2+ resulted in a recovery of MMP-2 activity, and furthermore, zinc-saturated LTF did not inhibit MMP-2. Together, these data strongly suggest that apolactoferrin is capable of removing the catalytic zinc from the active site of MMP-2, although an exosite-based interaction between the 2 proteins cannot be fully ruled out. This inhibitory activity suggests a novel function for LTF and may represent a novel regulatory mechanism that regulates proteolysis by MMP-2 in vivo.     

Molecular dynamics simulations of the unfolding mechanism of the catalytic domain from Aspergillus awamori var. X100 glucoamylase

In this study, 200 ps molecular dynamics simulations were conducted to investigate the unfolding mechanism of the catalytic domain of glucoamylase from Aspergillus awamori var. X100. The unfolding of this domain was suggested to follow a putative hierarchical manner, in which the heavily O-glycosylated belt region from residues T440 to A471 acted as the initiation site, followed by the alpha-helix secondary structure destruction, and then the collapse of the catalytic center pocket. The O-glycosylated belt region surrounded the surface of the catalytic domain in its native state at low temperature, whereas it was extended and is more suitable to be classified as part of the subsequent linker domain at high temperatures due to its high flexibility. The inner set helices of the (alpha/alpha)(6)-barrel seemed to exhibit higher helical content than the outer set ones at all temperatures examined. The distances between the C(alpha) of the three Cys residue pairs fluctuated rapidly at higher temperatures, indicating that these disulfide bonds have little effect on the structural stabilization. The melting temperature, at which the residual total helicity of the catalytic domain is 50%, is much lower than the critical temperature, at which the catalytic center pocket has lost its structural integrity.     

Evidence of the crucial role of the linker domain on the catalytic activity of human topoisomerase I by experimental and simulative characterization of the Lys681Ala mutant

The functional and structural-dynamical properties of the Lys681Ala mutation in the human topoisomerase IB linker domain have been investigated by catalytic assays and molecular dynamics simulation. The mutant is characterized by a comparable cleavage and a strongly reduced religation rate when compared to the wild type protein. The mutant also displays perturbed linker dynamics, as shown by analysis of the principal components of the motion, and a reduced electrostatic interaction with DNA. Inspection of the inter atomic distances in proximity of the active site shows that in the mutant the distance between the amino group of Lys532 side chain and the 5′ OH of the scissile phosphate is longer than the wild type enzyme, providing an atomic explanation for the reduced religation rate of the mutant. Taken together these results indicate the existence of a long range communication between the linker domain and the active site region and points out the crucial role of the linker in the modulation of the catalytic activity.