Mutational analysis of the hepatitis C virus RNA helicase.

The carboxyl-terminal three-fourths of the hepatitis C virus (HCV) NS3 protein has been shown to possess an RNA helicase activity, typical of members of the DEAD box family of RNA helicases. In addition, the NS3 protein contains four amino acid motifs conserved in DEAD box proteins. In order to inspect the roles of individual amino acid residues in the four conserved motifs (AXXXXGKS, DECH, TAT, and QRRGRTGR) of the NS3 protein, mutational analysis was used in this study. Thirteen mutant proteins were constructed, and their biochemical activities were examined. Lys1235 in the AXXXXGKS motif was important for basal nucleoside triphosphatase (NTPase) activity in the absence of polynucleotide cofactor. A serine in the X position of the DEXH motif disrupted the NTPase and RNA helicase activities. Alanine substitution at His1318 of the DEXH motif made the protein possess high NTPase activity. In addition, we now report inhibition of NTPase activity of NS3 by polynucleotide cofactor. Gln1486 was indispensable for the enzyme activity, and this residue represents a distinguishing feature between DEAD box and DEXH proteins. There are four Arg residues in the QRRGRTGR motif of the HCV NS3 protein, and the second, Arg1488, was important for RNA binding and enzyme activity, even though it is less well conserved than other Arg residues. Arg1490 and Arg1493 were essential for the enzymatic activity. As the various enzymatic activities were altered by mutation, the enzyme characteristics were also changed.     

Deep mutational scanning of an RRM domain of the Saccharomyces cerevisiae poly(A)-binding protein

The RNA recognition motif (RRM) is the most common RNA-binding domain in eukaryotes. Differences in RRM sequences dictate, in part, both RNA and protein-binding specificities and affinities. We used a deep mutational scanning approach to study the sequence-function relationship of the RRM2 domain of the Saccharomyces cerevisiae poly(A)-binding protein (Pab1). By scoring the activity of more than 100,000 unique Pab1 variants, including 1246 with single amino acid substitutions, we delineated the mutational constraints on each residue. Clustering of residues with similar mutational patterns reveals three major classes, composed principally of RNA-binding residues, of hydrophobic core residues, and of the remaining residues. The first class also includes a highly conserved residue not involved in RNA binding, G150, which can be mutated to destabilize Pab1. A comparison of the mutational sensitivity of yeast Pab1 residues to their evolutionary conservation reveals that most residues tolerate more substitutions than are present in the natural sequences, although other residues that tolerate fewer substitutions may point to specialized functions in yeast. An analysis of ∼40,000 double mutants indicates a preference for a short distance between two mutations that display an epistatic interaction. As examples of interactions, the mutations N139T, N139S, and I157L suppress other mutations that interfere with RNA binding and protein stability. Overall, this study demonstrates that living cells can be subjected to a single assay to analyze hundreds of thousands of protein variants in parallel.     

Directed evolution of alpha-aspartyl dipeptidase from Salmonella typhimurium.

Model-free approaches (error-prone PCR to introduce random mutations, DNA shuffling to combine positive mutations, and screening of the resultant mutant libraries) have been used to enhance the catalytic activity and thermostability of alpha-aspartyl dipeptidase from Salmonella typhimurium, which is uniquely able to hydrolyze Asp-X dipeptides (where X is any amino acid) and one tripeptide (Asp-Gly-Gly). Under double selective pressures of activity and thermostability, through two rounds of error-prone PCR and three sequential generations of DNA shuffling, coupled with screening, a mutant pepEM3074 with approximately 47-fold increased enzyme activity compared with its wild-type parent was obtained. Moreover, the stability of pepEM3074 is increased significantly. Three amino acid substitutions (Asn89His, Gln153Glu, and Leu205Arg), two of them are near the active site and substrate binding pocket, were identified by sequencing the genes encoding this evolved enzyme. The mechanism of the enhancement of activity and stability was analyzed in this paper.     

Defining the active site of Schizosaccharomyces pombe C-terminal domain phosphatase Fcp1

Fcp1 is an essential protein serine phosphatase that dephosphorylates the C-terminal domain (CTD) of RNA polymerase II. By testing the effects of serial N- and C-terminal deletions of the 723-amino acid Schizosaccharomyces pombe Fcp1, we defined a minimal phosphatase domain spanning amino acids 156-580. We employed site-directed mutagenesis (introducing 24 mutations at 14 conserved positions) to locate candidate catalytic residues. We found that alanine substitutions for Arg(223), Asp(258), Lys(280), Asp(297), and Asp(298) abrogated the phosphatase activity with either p-nitrophenyl phosphate or CTD-PO(4) as substrates. Structure-activity relationships were determined by introducing conservative substitutions at each essential position. Our results, together with previous mutational studies, highlight a constellation of seven amino acids (Asp(170), Asp(172), Arg(223), Asp(258), Lys(280), Asp(297), and Asp(298)) that are conserved in all Fcp1 orthologs and likely comprise the active site. Five of these residues (Asp(170), Asp(172), Lys(280), Asp(297), and Asp(298)) are conserved at the active site of T4 polynucleotide 3'-phosphatase, suggesting that Fcp1 and T4 phosphatase are structurally and mechanistically related members of the DXD phosphotransferase superfamily.     

Mutational analysis of the guanylyltransferase component of Mammalian mRNA capping enzyme

RNA guanylyltransferase is an essential enzyme that catalyzes the second of three steps in the synthesis of the 5'-cap structure of eukaryotic mRNA. Here we conducted a mutational analysis of the guanylyltransferase domain of the mouse capping enzyme Mce1. We introduced 50 different mutations at 22 individual amino acids and assessed their effects on Mce1 function in vivo in yeast. We identified 16 amino acids as being essential for Mce1 activity (Arg299, Arg315, Asp343, Glu345, Tyr362, Asp363, Arg380, Asp438, Gly439, Lys458, Lys460, Asp468, Arg530, Asp532, Lys533, and Asn537) and clarified structure-activity relationships by testing the effects of conservative substitutions. The new mutational data for Mce1, together with prior mutational studies of Saccharomyces cerevisiae guanylyltransferase and the crystal structures of Chlorella virus and Candida albicans guanylyltransferases, provide a coherent picture of the functional groups that comprise and stabilize the active site. Our results extend and consolidate the hypothesis of a shared structural basis for catalysis by RNA capping enzymes, DNA ligases, and RNA ligases, which comprise a superfamily of covalent nucleotidyl transferases defined by a constellation of conserved motifs. Analysis of the effects of motif VI mutations on Mce1 guanylyltransferase activity in vitro highlights essential roles for Arg530, Asp532, Lys533, and Asn537 in GTP binding and nucleotidyl transfer.     

Identification of key residues in the amino-terminal third of human interleukin-1 alpha

Two mutational approaches were used to perform a thorough structure-function analysis of the first 53 residues of the 159-residue cytokine human interleukin-1 alpha (hIL-1 alpha). In this study, a total of 26 deletions, 97 multiple amino acid substitutions, and 46 single amino acid substitutions were examined. A synthetic hIL-1 alpha gene with many unique restriction sites was constructed to facilitate the molecular manipulations that were performed. The mutational methods employed include: Bal-31 exonuclease-generated deletions at unique restriction sites and combinatorial cassette mutagenesis via segment replacement with synthetic DNA. The mutant hIL-1 alpha proteins were expressed at high levels in Escherichia coli and were assayed for biological activity in a mouse T cell proliferation assay. We observed that the activity of hIL-1 alpha was extraordinarily sensitive to deletion mutations. Most internal deletions of as few as 1 or 2 residues substantially reduced biological activity. Combinatorial cassette mutagenesis on residues 13-53 of hIL-1 alpha identified 15 important residue positions. Of these, 8 displayed strong preferences for residues with hydrophilic side chains, and the remainder preferred hydrophobic side chains. We found that functional hIL-1 alpha had an absolute requirement for a basic residue (Arg, Lys, or His) at either position 15 or 16, and that Leu was preferred at position 40.     

Leukotriene A4 hydrolase, insights into the molecular evolution by homology modeling and mutational analysis of enzyme from Saccharomyces cerevisiae

Mammalian leukotriene A4 (LTA4) hydrolase is a bifunctional zinc metalloenzyme possessing an Arg/Ala aminopeptidase and an epoxide hydrolase activity, which converts LTA4 into the chemoattractant LTB4. We have previously cloned an LTA4 hydrolase from Saccharomyces cerevisiae with a primitive epoxide hydrolase activity and a Leu aminopeptidase activity, which is stimulated by LTA4. Here we used a modeled structure of S. cerevisiae LTA4 hydrolase, mutational analysis, and binding studies to show that Glu-316 and Arg-627 are critical for catalysis, allowing us to a propose a mechanism for the epoxide hydrolase activity. Guided by the structure, we engineered S. cerevisiae LTA4 hydrolase to attain catalytic properties resembling those of human LTA4 hydrolase. Thus, six consecutive point mutations gradually introduced a novel Arg aminopeptidase activity and caused the specific Ala and Pro aminopeptidase activities to increase 24 and 63 times, respectively. In contrast to the wild type enzyme, the hexuple mutant was inhibited by LTA4 for all tested substrates and to the same extent as for the human enzyme. In addition, these mutations improved binding of LTA4 and increased the relative formation of LTB4, whereas the turnover of this substrate was only weakly affected. Our results suggest that during evolution, the active site of an ancestral eukaryotic zinc aminopeptidase has been reshaped to accommodate lipid substrates while using already existing catalytic residues for a novel, gradually evolving, epoxide hydrolase activity. Moreover, the unique ability to catalyze LTB4 synthesis appears to be the result of multiple and subtle structural rearrangements at the catalytic center rather than a limited set of specific amino acid substitutions.     

Complete amino acid sequence of three reptile lysozymes

To study the structure and function of reptile lysozymes, we have reported their purification, and in this study we have established the amino acid sequence of three egg white lysozymes in soft-shelled turtle eggs (SSTL A and SSTL B from Trionyx sinensis, ASTL from Amyda cartilaginea) by using the rapid peptide mapping method. The established amino acid sequence of SSTL A, SSTL B, and ASTL showed substitutions of 43, 42, and 44 residues respectively when compared with the HEWL (hen egg white lysozyme) sequence. In these reptile lysozymes, SSTL A had one substitution compared with SSTL B (Gly126Asp) and had an N-terminal extra Gly and 11 substitutions compared with ASTL. SSTL B had an N-terminal extra Gly and 10 residues different from ASTL. The sequence of SSTL B was identical to soft-shelled turtle lysozyme from STL (Trionyx sinensis japonicus). The Ile residue at position 93 of ASTL is the first report in all C-type lysozymes. Furthermore, amino acid substitutions (Phe34His, Arg45Tyr, Thr47Arg, and Arg114Tyr) were also found at subsites E and F when compared with HEWL. The time course using N-acetylglucosamine pentamer as a substrate exhibited a reduction of the rate constant of glycosidic cleavage and increase of binding free energy for subsites E and F, which proved the contribution for amino acids mentioned above for substrate binding at subsites E and F. Interestingly, the variable binding free energy values occurred on ASTL, may be contributed from substitutions at outside of subsites E and F.     

An ultra‐high affinity protein–protein interface displaying sequence‐robustness

Protein–protein interactions are crucial in biology and play roles in for example, the immune system, signaling pathways, and enzyme regulation. Ultra‐high affinity interactions (K _d <0.1 nM) occur in these systems, however, structures and energetics behind stability of ultra‐high affinity protein–protein complexes are not well understood. Regulation of the starch debranching barley limit dextrinase (LD) and its endogenous cereal type inhibitor (LDI) exemplifies an ultra‐high affinity complex (K _d of 42 pM). In this study the LD–LDI complex is investigated to unveil how robust the ultra‐high affinity is to LDI sequence variation at the protein–protein interface and whether alternative sequences can retain the ultra‐high binding affinity. The interface of LD–LDI was engineered using computational protein redesign aiming at identifying LDI variants predicted to retain ultra‐high binding affinity. These variants present a very diverse set of mutations going beyond conservative and alanine substitutions typically used to probe interfaces. Surface plasmon resonance analysis of the LDI variants revealed that high affinity of LD–LDI requires interactions of several residues at the rim of the protein interface, unlike the classical hotspot arrangement where key residues are found at the center of the interface. Notably, substitution of interface residues in LDI, including amino acids with functional groups different from the wild‐type, could occur without loss of affinity. This demonstrates that ultra‐high binding affinity can be conferred without hotspot residues, thus making complexes more robust to mutational drift in evolution. The present mutational analysis also demonstrates how energetic coupling can emerge between residues at large distances at the interface.     

The Amino Acid Sequence of Monal Pheasant Lysozyme and Its Activity

The amino acid sequence of monal pheasant lysozyme and its activity were analyzed. Carboxymethylated lysozyme was digested with trypsin and the resulting peptides were sequenced. The established amino acid sequence had one amino acid substitution at position 102 (Arg to Gly) comparing with Indian peafowl lysozyme and four amino acid substitutions at positions 3 (Phe to Tyr), 15 (His to Leu), 41 (Gln to His), and 121 (Gln to His) with chicken lysozyme. Analysis of the time-courses of reaction using N-acetylglucosamine pentamer as a substrate showed a difference of binding free energy change (-0.4 kcal/mol) at subsites A between monal pheasant and Indian peafowl lysozyme. This was assumed to be caused by the amino acid substitution at subsite A with loss of a positive charge at position 102 (Arg102 to Gly).     

Systematic mutation of bacteriophage T4 lysozyme

Amber mutations were introduced into every codon (except the initiating AUG) of the bacteriophage T4 lysozyme gene. The amber alleles were introduced into a bacteriophage P22 hybrid, called P22 e416, in which the normal P22 lysozyme gene is replaced by its T4 homologue, and which consequently depends upon T4 lysozyme for its ability to form a plaque. The resulting amber mutants were tested for plaque formation on amber suppressor strains of Salmonella typhimurium. Experiments with other hybrid phages engineered to produce different amounts of wild-type T4 lysozyme have shown that, to score as deleterious, a mutation must reduce lysozyme activity to less than 3% of that produced by wild-type P22 e416. Plating the collection of amber mutants covering 163 of the 164 codons of T4 lysozyme, on 13 suppressor strains that each insert a different amino acid substitutions at every position in the protein (except the first). Of the resulting 2015 single amino acid substitutions in T4 lysozyme, 328 were found to be sufficiently deleterious to inhibit plaque formation. More than half (55%) of the positions in the protein tolerated all substitutions examined. Among (N-terminal) amber fragments, only those of 161 or more residues are active. The effects of many of the deleterious substitutions are interpretable in light of the known structure of T4 lysozyme. Residues in the molecule that are refractory to replacements generally have solvent-inaccessible side-chains; the catalytic Glu11 and Asp20 residues are notable exceptions. Especially sensitive sites include residues involved in buried salt bridges near the catalytic site (Asp10, Arg145 and Arg148) and a few others that may have critical structural roles (Gly30, Trp138 and Tyr161).     

Long range effects of amino acid substitutions in the catalytic chain of aspartate transcarbamoylase. Localized replacements in the carboxyl-terminal alpha-helix cause marked alterations in allosteric properties and intersubunit interactions.

A single alpha-helical polypeptide segment of 21 amino acids near the carboxyl terminus of the catalytic chain of aspartate transcarbamoylase from Escherichia coli has been shown recently to be important for the in vivo folding of the chains and assembly of the enzyme (Peterson, C. B., and Schachman, H. K. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 458-462). Calorimetric measurements on purified mutant enzymes showed that single amino acid replacements within this secondary structural element affect the overall thermal stability of the oligomeric enzyme and the energetics of the interactions between polypeptide chains within the holoenzyme. Studies presented here demonstrate that marked changes in cooperativity occur due to single amino acid substitutions. Replacement of Gln288 by either Ala or Glu leads to a striking increase in the Hill coefficient of the holoenzymes and a substantial increase in the aspartate concentration corresponding to one-half Vmax. In contrast, the isolated catalytic trimers harboring these same substitutions were similar in activity to the wild-type subunit, with the same affinity for aspartate as indicated by the values of Km. Substituting Ala for the only charged residue in the helix, Arg296, caused a marked reduction in enzyme activity, as well as a greatly reduced stability of the holoenzyme due to a substantial weakening of the interactions between the catalytic and regulatory subunits. A subunit exchange method was used to demonstrate the changes in interchain interactions resulting from the amino acid substitutions and to show the additional weakening upon the binding of the bisubstrate ligand, N-(phosphonacetyl)-L-aspartate, at the active sites. Taken together, the results on this series of mutant enzymes illustrate how the effects of single amino acid replacements in one element of secondary structure are propagated throughout the molecule to positions remote from the site of the substitution.     

Mutational analysis of vaccinia virus topoisomerase identifies residues involved in DNA binding.

Vaccinia DNA topoisomerase catalyzes the cleavage and re-joining of DNA strands through a DNA-(3'-phosphotyrosyl)-enzyme intermediate formed at a specific target sequence, 5'-(C/T)CCTT downward arrow. The 314 aa protein consists of three protease-resistant structural domains demarcated by protease-sensitive interdomain segments referred to as the bridge and the hinge. The bridge is defined by trypsin-accessible sites at Arg80, Lys83 and Arg84. Photocrosslinking and proteolytic footprinting experiments suggest that residues near the interdomain bridge interact with DNA. To assess the contributions of specific amino acids to DNA binding and transesterification chemistry, we introduced alanine substitutions at 16 positions within a 24 aa segment from residues 63 to 86(DSKGRRQYFYGKMHVQNRNAKRDR). Assays of the rates of DNA relaxation under conditions optimal for the wild-type topoisomerase revealed significant mutational effects at six positions; Arg67, Tyr70, Tyr72, Arg80, Arg84 and Asp85. The mutated proteins displayed normal or near-normal rates of single-turnover transesterification to DNA. The effects of amino acid substitutions on DNA binding were evinced by inhibition of covalent adduct formation in the presence of salt and magnesium. The mutant enzymes also displayed diminished affinity for a subset of cleavage sites in pUC19 DNA. Tyr70 and Tyr72 were subjected to further analysis by replacement with Phe, His, Gln and Arg. At both positions, the aromatic moiety was important for DNA binding.     

Structure/activity relationships of C-terminal neuropeptide Y peptide segments and analogues composed of sequence 1-4 linked to 25-36

C-terminal analogues of neuropeptide Y have been synthesized. The influence of chain length, single-amino-acid substitutions and segment substitutions on receptor binding, biological activity and conformational properties has been investigated. Receptor binding and in vivo assays revealed biological activity already for amino acids 28-36 of neuropeptide Y [neuropeptide Y-(Ac-28-36)-peptide] which increased with increasing chain length. Replacement of Arg25 in neuropeptide Y-(Ac-25-36)-peptide had no influence on binding, whereas Arg33 and Arg35 cannot be replaced by lysine or ornithine without considerable decrease in receptor binding. The introduction of conformational constraints by the 2-aminoisobutyric acid residue (Aib) in position 30 and replacing the amino acids 28-32 by Ala-Aib-Ala-Aib-Ala decreased receptor binding. However, the corresponding Aib-Ala-Aib-Ala-Aib-substituted analogue and a more flexible analogue with Gly5 at position 28-32 exhibited considerable affinity for the receptor. All these substitutions led to a decrease in postsynaptic activity. Strong agonistic activities could be detected in a series of 10 discontinuous analogues, which are constructs of N-terminal parts linked via different spacer molecules to C-terminal segments. One of the most active molecules was neuropeptide Y amino acids 1-4 linked to amino acids 25-36 through aminohexanoic acid (Ahx) [neuropeptide Y-(1-4-Ahx-25-36)-peptide].     

Combining Natural Sequence Variation with High Throughput Mutational Data to Reveal Protein Interaction Sites

Many protein interactions are conserved among organisms despite changes in the amino acid sequences that comprise their contact sites, a property that has been used to infer the location of these sites from protein homology. In an inter-species complementation experiment, a sequence present in a homologue is substituted into a protein and tested for its ability to support function. Therefore, substitutions that inhibit function can identify interaction sites that changed over evolution. However, most of the sequence differences within a protein family remain unexplored because of the small-scale nature of these complementation approaches. Here we use existing high throughput mutational data on the in vivo function of the RRM2 domain of the Saccharomyces cerevisiae poly(A)-binding protein, Pab1, to analyze its sites of interaction. Of 197 single amino acid differences in 52 Pab1 homologues, 17 reduce the function of Pab1 when substituted into the yeast protein. The majority of these deleterious mutations interfere with the binding of the RRM2 domain to eIF4G1 and eIF4G2, isoforms of a translation initiation factor. A large-scale mutational analysis of the RRM2 domain in a two-hybrid assay for eIF4G1 binding supports these findings and identifies peripheral residues that make a smaller contribution to eIF4G1 binding. Three single amino acid substitutions in yeast Pab1 corresponding to residues from the human orthologue are deleterious and eliminate binding to the yeast eIF4G isoforms. We create a triple mutant that carries these substitutions and other humanizing substitutions that collectively support a switch in binding specificity of RRM2 from the yeast eIF4G1 to its human orthologue. Finally, we map other deleterious substitutions in Pab1 to inter-domain (RRM2–RRM1) or protein-RNA (RRM2–poly(A)) interaction sites. Thus, the combined approach of large-scale mutational data and evolutionary conservation can be used to characterize interaction sites at single amino acid resolution.     

Two Amino Acid Residues Confer Different Binding Affinities of Abelson Family Kinase Src Homology 2 Domains for Phosphorylated Cortactin

The closely related Abl family kinases, Arg and Abl, play important non-redundant roles in the regulation of cell morphogenesis and motility. Despite similar N-terminal sequences, Arg and Abl interact with different substrates and binding partners with varying affinities. This selectivity may be due to slight differences in amino acid sequence leading to differential interactions with target proteins. We report that the Arg Src homology (SH) 2 domain binds two specific phosphotyrosines on cortactin, a known Abl/Arg substrate, with over 10-fold higher affinity than the Abl SH2 domain. We show that this significant affinity difference is due to the substitution of arginine 161 and serine 187 in Abl to leucine 207 and threonine 233 in Arg, respectively. We constructed Abl SH2 domains with R161L and S187T mutations alone and in combination and find that these substitutions are sufficient to convert the low affinity Abl SH2 domain to a higher affinity “Arg-like” SH2 domain in binding to a phospho-cortactin peptide. We crystallized the Arg SH2 domain for structural comparison to existing crystal structures of the Abl SH2 domain. We show that these two residues are important determinants of Arg and Abl SH2 domain binding specificity. Finally, we expressed Arg containing an “Abl-like” low affinity mutant Arg SH2 domain (L207R/T233S) and find that this mutant, although properly localized to the cell periphery, does not support wild type levels of cell edge protrusion. Together, these observations indicate that these two amino acid positions confer different binding affinities and cellular functions on the distinct Abl family kinases.     

Functional synergism between the most common polymorphism in human alanine:glyoxylate aminotransferase and four of the most common disease-causing mutations

The autosomal recessive disorder primary hyperoxaluria type 1 (PH1) is caused by a deficiency of the liver-specific pyridoxal-phosphate-dependent enzyme alanine:glyoxylate aminotransferase (AGT). Numerous mutations and polymorphisms in the gene encoding AGT have been identified, but in only a few cases has the causal relationship between genotype and phenotype actually been demonstrated. In this study, we have determined the effects of the most common naturally occurring amino acid substitutions (both normal polymorphisms and disease-causing mutations) on the properties, especially specific catalytic activity, of purified recombinant AGT. The results presented in this paper show the following: 1) normal human His-tagged AGT can be expressed at high levels in Escherichia coli and purified in a correctly folded, dimerized and catalytically active state; 2) presence of the common P11L polymorphism decreases the specific activity of purified recombinant AGT by a factor of three; 3) AGTs containing four of the most common PH1-specific mutations (G41R, F152I, G170R, and I244T) are all soluble and catalytically active in the absence of the P11L polymorphism, but in its presence all lead to protein destabilization and aggregation into inclusion bodies; 4) naturally occurring and artificial amino acid substitutions that lead to peroxisome-to-mitochondrion AGT mistargeting in mammalian cells also lead to destabilization and aggregation in E. coli; and 5) the PH1-specific G82E mutation abolishes AGT catalytic activity by interfering with cofactor binding, as does the artificial K209R mutation at the putative site of cofactor Shiff base formation. These results are discussed in the light of the high allelic frequency ( approximately 20%) of the P11L polymorphism and its importance in determining the phenotypic manifestations of mutations in PH1.     

Characterization of Novel MSX1 Mutations Identified in Japanese Patients with Nonsyndromic Tooth Agenesis

Since MSX1 and PAX9 are linked to the pathogenesis of nonsyndromic tooth agenesis, we performed detailed mutational analysis of these two genes sampled from Japanese patients. We identified two novel MSX1 variants with an amino acid substitution within the homeodomain; Thr174Ile (T174I) from a sporadic hypodontia case and Leu205Arg (L205R) from a familial oligodontia case. Both the Thr174 and Leu205 residues in the MSX1 homeodomain are highly conserved among different species. To define possible roles of mutations at these amino acids in the pathogenesis of nonsyndromic tooth agenesis, we performed several functional analyses. It has been demonstrated that MSX1 plays a pivotal role in hard tissue development as a suppressor for mesenchymal cell differentiation. To evaluate the suppression activity of the variants in mesenchymal cells, we used the myoD-promoter, which is one of convenient reporter assay system for MSX1. Although the gene products of these MSX1 variants are stable and capable of normal nuclear localization, they do not suppress myoD-promoter activity in differentiated C2C12 cells. To clarify the molecular mechanisms underlying our results, we performed further analyses including electrophoretic mobility shift assays, and co-immunoprecipitation assays to survey the molecular interactions between the mutant MSX1 proteins and the oligonucleotide DNA with MSX1 consensus binding motif or EZH2 methyltransferase. Since EZH2 is reported to interact with MSX1 and regulate MSX1 mediated gene suppression, we hypothesized that the T174I and L205R substitutions would impair this interaction. We conclude from the results of our experiments that the DNA binding ability of MSX1 is abolished by these two amino acid substitutions. This illustrates a causative role of the T174I and L205R MSX1 homeodomain mutations in tooth agenesis, and suggests that they may influence cell proliferation and differentiation resulting in lesser tooth germ formation in vivo.     

Antithrombin III Basel. Identification of a Pro-Leu substitution in a hereditary abnormal antithrombin with impaired heparin cofactor activity

Antithrombin III Basel is a hereditary abnormal antithrombin with normal progressive inhibition activity (normal reactive site) and reduced heparin cofactor activity (impaired heparin binding site). Structures of antithrombin III Basel and normal antithrombin III isolated from the same patient were compared by peptide mapping using the dimethylaminoazobenzene isothiocyanate precolumn derivatization technique. Of the approximately 50 tryptic peptides of normal and abnormal antithrombin III, one peptide comprising residues 40-46 had a different retention time in reversed-phase high performance liquid chromatography. The amino acid sequence of the peptide from antithrombin III Basel had a single substitution of Pro (normal) by Leu (abnormal) at position 41. This substitution is close to an Arg (residue 47) and a Trp (residue 49) which have previously been shown to be critical for heparin binding by antithrombin III. Although additional amino acid substitutions in antithrombin III Basel cannot be ruled out, this Pro-Leu replacement could cause a conformational change by increasing both the helical structure and the hydrophobicity around residue 41. These data suggest that: (i) the heparin binding site of antithrombin III encompasses the region containing residues 41, 47, and 49; and (ii) the impaired heparin cofactor activity of antithrombin III Basel is likely due to a conformational change of the heparin binding site induced by the Pro-Leu substitution at position 41.     

Estimation of the frequency of amino acid substitutions causing instability of the spatial structure in an overall mutation spectrum of alpha- and beta-subunits of human hemoglobin

The influence of single amino acid substitutions on the stability of alpha- and beta-chains of human hemoglobin was investigated by the computer method. The method was based on characteristics of protein tertiary structure and physico-chemical properties of amino acids. Frequencies of unstable mutations in the total mutational spectra of alpha- and beta-subunits of human hemoglobin were analysed: instability was produced by 26% of mutations in the alpha-subunit and by 32% in the beta-subunit. These results support the idea that certain limitations exist for stability changes produced by amino acid substitutions.