A study of roles of evolutionarily invariant proline 30 and glycine 34 of cytochrome c

The previous studies (Juillerat, M. A., and Taniuchi, H. (1986) J. Biol. Chem. 261, 2697-2711), using a three-fragment complex (1-25)H X (28-38) X (39-104) of horse cytochrome c, have shown that invariant leucine 32 and partially invariant leucine 35, both buried in the interior, exhibit a striking difference in perturbation of binding fragment (28-38) by substitution with isoleucine. Then the idea has been proposed that the energy states of leucine 32, the Met-80-S-heme-Fe bond and other distant residues such as tryptophan 59 would be coupled to generate extra force while leucine 35 would be less important for such coupling if it were involved. In the present studies we synthesized three (28-38) analogs substituting invariant proline 30 with glycine or invariant glycine 34 with alanine or serine. Thermodynamic and kinetic studies and UV CD and biological activity measurements were carried out on binding of the analogs to complex (1-25)H X (39-104). The results with the ferric form show that perturbations of delta G, delta H, and delta S associated with formation of the intermediate complex and with the ensuing process by the Gly34----Ala or Ser substitution result in weakening the Met-80-S-heme-Fe bond formed in the latter process; in contrast, perturbation by the Pro30----Gly substitution is small. However, the biological activity is more perturbed by the Pro30----Gly substitution than by the Gly34----Ala or Ser; and in the Gly34----Ala or Ser substitution the complex appears to be more readily activated for both formation and disruption of the Met-80-S-heme-Fe bond at 20 degrees C and below than without substitution. In all cases reduction of the heme strengthens the binding of fragment (28-38). However, striking are the increases in perturbation (less negative) of both delta H and delta S for binding of fragment (28-38) to form the ground state on reduction of the heme in the Pro30----Gly, Gly34----Ala or Ser (the present studies), and Leu32----norvaline (the previous studies) substitutions. It is known that fluctuation of the atomic positions of most residues of tuna ferrocytochrome c including Pro30, Leu32, and Gly34 increases on oxidation of the heme and that these three residues are among those showing the least fluctuating atomic positions (Takano, T., and Dickerson, R.E. (1982) in Electron Transport and Oxygen Utilization (Ho, C., ed) pp. 17-26, Elsevier/North-Holland Biomedical Press, New York).(ABSTRACT TRUNCATED AT 400 WORDS)     

Transformation of the mechanism of triple-helix peptide folding in the absence of a C-terminal nucleation domain and its implications for mutations in collagen disorders

Folding abnormalities of the triple helix have been demonstrated in collagen diseases such as osteogenesis imperfecta in which the mutation leads to the substitution of a single Gly in the (Gly-X-Y)n sequence pattern by a larger residue. Model peptides can be used to clarify the details of normal collagen folding and the consequences of the interruption of that folding by a Gly substitution. NMR and CD studies show that placement of a (GPO)4 nucleation domain at the N terminus rather than the C terminus of a native collagen sequence allows the formation of a stable triple helix but alters the folding mechanism. Although C- to N-terminal directional folding occurs when the nucleation domain is at the C terminus, there is no preferential folding direction when the nucleation domain is at the N terminus. The lack of zipper-like directional folding does not interfere with triple-helix formation, and when a Gly residue is replaced by Ser to model an osteogenesis imperfecta mutation, the peptide with the N-terminal (GPO)4 domain can still form a good triple helix N-terminal to the mutation site. These peptide studies raise the possibility that mutant collagen could fold in a C to N direction in a zipper-like manner up to the mutation site and that completion of the triple helix N-terminal to the mutation would involve an alternative mechanism.     

Functional consequences of alterations to Gly310, Gly770, and Gly801 located in the transmembrane domain of the Ca(2+)-ATPase of sarcoplasmic reticulum.

Site-specific mutagenesis was used to replace Gly310, Gly770, and Gly801, located in the transmembrane domain of the sarcoplasmic reticulum Ca(2+)-ATPase, with either alanine or valine. In addition, Gly310 was substituted with proline. In the Gly310----Ala mutant, the Vmax for Ca2+ transport and ATPase activity was reduced to about 40% of the wild type activity, but the apparent Ca2+ affinity was close to normal. The Gly310----Val and Gly310----Pro mutants were devoid of Ca2+ transport or ATPase activity and displayed more than a 20-fold reduction in the apparent Ca2+ affinities measured in the phosphorylation assays with either ATP or Pi. In these mutants, the rate of phosphoenzyme hydrolysis was reduced, and the ADP-insensitive phosphoenzyme intermediate accumulated. The apparent affinity for Pi was increased in the absence, but not in the presence, of dimethyl sulfoxide. The properties of this new class of Ca(2+)-ATPase mutants ("E2/E2P" type) are consistent with a conformational state in which the protein-phosphate interaction is stabilized and the Ca(2+)-protein interaction is destabilized. The Gly770----Ala mutant transported Ca2+ with a Vmax close to that of the wild type, but displayed more than a 20-fold reduction of apparent Ca2+ affinity. The Gly770----Val mutant was not phosphorylated from either ATP or Pi. The Gly801----Ala mutant transported Ca2+ with a Vmax of 126% that of the wild type, hydrolyzed ATP at the same Vmax as the wild type in the presence of calcium ionophore, and displayed a 3-fold reduction in apparent Ca2+ affinity. The Gly801----Val mutant was unable to transport Ca2+ and to be phosphorylated from ATP, even at a Ca2+ concentration of 1 mM, but Ca2+ in the micromolar range inhibited phosphorylation from Pi. The ability to bind ATP with normal affinity was retained. The properties of this mutant are consistent with a disruption of one of the two Ca2+ binding sites required for phosphorylation with ATP.     

Folding and conformational consequences of glycine to alanine replacements at different positions in a collagen model peptide

The collagen model peptide T1-892 includes a C-terminal nucleation domain, (Gly-Pro-Hyp)(4), and an N-terminal (Gly-X-Y)(6) sequence taken from type I collagen. In osteogenesis imperfecta (OI) and other collagen diseases, single base mutations often convert one Gly to a larger residue, and T1-892 homologues modeling such mutations were synthesized with Gly to Ala substitutions in either the (Gly-Pro-Hyp)(4) domain, Gly25Ala, or the (Gly-X-Y)(6) domain, Gly10Ala. CD and NMR studies show the Gly10Ala peptide forms a normal triple-helix at the C-terminal end and propagates from the C- to the N-terminus until the Gly --> Ala substitution is encountered. At this point, triple-helix folding is terminated and cannot be reinitiated, leaving a nonhelical N-terminus. A decreased thermal stability is observed as a result of the shorter length of the triple-helix. In contrast, introduction of the Gly to Ala replacement at position 25, in the nucleation domain, shifts the monomer/trimer equilibrium toward the monomer form. The increased monomer and lower trimer populations are reflected in the dramatic decrease in triple-helix content and stability. Unlike the Ala replacement at position 10, the Ala substitution in the (Gly-Pro-Hyp)(4) region can still be incorporated into a triple-helix, but at a greatly decreased rate of folding, since the original efficient nucleation site is no longer operative. The specific consequences of Gly to Ala replacements in two distinctive sequences in this triple-helical peptide may help clarify the variability in OI clinical severity resulting from mutations at different sites along type I collagen chains.     

Importance of domain closure for homotropic cooperativity in Escherichia coli aspartate transcarbamylase

The importance of the interdomain bridging interactions observed only in the R-state structure of Escherichia coli aspartate transcarbamylase between Glu-50 of the carbamoyl phosphate domain with both Arg-167 and Arg-234 of the aspartate domain has been investigated by using site-specific mutagenesis. Two mutant versions of aspartate transcarbamylase were constructed, one with alanine at position 50 (Glu-50----Ala) and the other with aspartic acid at position 50 (Glu-50----Asp). The alanine substitution totally prevents the interdomain bridging interactions, while the aspartic acid substitution was expected to weaken these interactions. The Glu-50----Ala holoenzyme exhibits a 15-fold loss of activity, no substrate cooperativity, and a more than 6-fold increase in the aspartate concentration at half the maximal observed specific activity. The Glu-50----Asp holoenzyme exhibits a less than 3-fold loss of activity, reduced cooperativity for substrates, and a 2-fold increase in the aspartate concentration at half the maximal observed specific activity. Although the Glu-50----Ala enzyme exhibits no homotropic cooperativity, it is activated by N-(phosphonoacetyl)-L-aspartate (PALA). As opposed to the wild-type enzyme, the Glu-50----Ala enzyme is activated by PALA at saturating concentrations of aspartate. At subsaturating concentrations of aspartate, both mutant enzymes are activated by ATP, but are inhibited less by CTP than is the wild-type enzyme. At saturating concentrations of aspartate, the Glu-50----Ala enzyme is activated by ATP and inhibited by CTP to an even greater extent than at subsaturating concentrations of aspartate.(ABSTRACT TRUNCATED AT 250 WORDS)     

Analysis of fibrinogen A alpha-fusion proteins. Mutants which inhibit thrombin equivalently are not equally good substrates

We have examined the interaction of thrombin with fibrinogen A alpha chain residues 7-16. Using genetically engineered constructions, we have synthesized in Escherichia coli a fibrinogen A alpha 1-50 fusion protein and seven mutant proteins with single amino acid substitutions. These are: Asp7----Ala, Phe8----Tyr, Glu11----Ala, Gly12----Val, Gly13----Val, Gly14----Val, and Arg16----Leu. Competitive immunoassay of cell lysates showed that all the mutations but one, Arg16----Leu, altered the structure of the protein such that cross-reactivity with the A alpha-specific monoclonal antibody, Y18, was significantly reduced. The fusion proteins were purified and analyzed as thrombin inhibitors and substrates. All the fusion proteins are competitive inhibitors of the amidolytic hydrolysis of Spectrozyme TH, a thrombin-specific chromogenic substrate, with inhibition constants corresponding to that for fibrinogen. We conclude that these 7 amino acid substitutions do not alter thrombin binding to the fusion proteins. The fusion proteins were tested as substrates by monitoring thrombin-dependent peptide release. The natural sequence and three mutants, Asp7----Ala, Glu11----Ala, and Gly14----Val, are good substrates. The other mutants are either poor substrates or are not cleaved by thrombin within A alpha 1-50. These results indicate that residues between Asp7 and Arg16 are critical to efficient peptide hydrolysis, whereas residues outside this region are critical to thrombin binding.     

Site-directed mutagenesis in rat liver 6-phosphofructo-2-kinase. Mutation at the fructose 6-phosphate binding site affects phosphate activation.

To identify those residues involved in fructose 6-phosphate binding to the kinase domain of rat liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase site-directed mutations were engineered at Lys194, Arg195, Arg230, and Arg238. The mutant enzymes were purified to homogeneity by anion exchange and Blue-Sepharose chromatography and/or substrate elution from phosphocellulose columns. Circular dichroism experiments demonstrated that all of the single amino acid mutations had no effect on the secondary structure of the protein. In addition, when fructose-2,6-bisphosphatase activity was measured, all mutants had Km values for fructose 2,6-bisphosphate, Ki values for fructose 6-phosphate, and maximal velocities similar to that of the wild-type enzyme. Mutation of Arg195----Ala, or His, had little or no effect on the maximal velocity of the kinase but increased the Km for fructose 6-phosphate greater than 3,000-fold. Furthermore, the Ka for phosphate for Arg195Ala was increased 100-fold compared with the wild-type enzyme. Mutation of Lys194----Ala had no effect on maximal velocity or the Km for fructose 6-phosphate. Mutation of either Arg230 or Arg238----Ala increased the maximal velocity and the Km for fructose-6 phosphate of the kinase by 2-3-fold but had no effect on fructose-2,6-bisphosphatase. However, the Km values for ATP of the Arg230Ala and Arg238Ala mutants were 30-40-fold higher than that for the wild-type enzyme. Mutation of Gly48----Ala resulted in a form with no kinase activity, but fructose-2,6-bisphosphatase activity was identical to that of the wild-type enzyme. The results indicate that: 1) Arg195 is a critical residue for the binding of fructose 6-phosphate to the 6-phospho-fructo-2-kinase domain, and that interaction of the sugar phosphate with Arg195 is highly specific since mutation of the adjacent Lys194----Ala had no effect on fructose 6-phosphate binding; 2) Arg195 also play an important role in the binding of inorganic phosphate; and 3) Gly48 is an important residue in the nucleotide binding fold of 6-phosphofructo-2-kinase and that both Arg230 and Arg238 are also involved in ATP binding; and 4) the bifunctional enzyme has two separate and independent fructose 6-phosphate binding sites.     

Nuclear magnetic resonance characterization of peptide models of collagen-folding diseases

Misfolding of the triple helix has been shown to play a critical role in collagen diseases. The substitution of a single Gly by another amino acid breaks the characteristic repeating (Gly-X-Y)n sequence pattern and results in connective tissue disease such as osteogenesis imperfecta. Nuclear magnetic resonance (NMR) studies of normal and mutated collagen triple-helical peptides offer an opportunity to characterize folding and conformational alterations at the substitution site, as well as at positions upstream and downstream of a Gly mutation. The NMR studies suggest that the local sequences surrounding the substitution site, and the renucleation sequences N-terminal to and adjacent to the substitution site, may be critical in defining the clinical phenotype of osteogenesis imperfecta. These studies may pave the way to understanding the mechanism by which a single Gly substitution in collagen can lead to pathological conditions.     

Structure-function studies on bacteriorhodopsin. VIII. Substitutions of the membrane-embedded prolines 50, 91, and 186: the effects are determined by the substituting amino acids

To study their role in the structure and function of bacteriorhodopsin, three prolines, presumed to be in the membrane-embedded alpha-helices, have been individually replaced as follows: Pro-50 and Pro-91 each by Gly and Ala and Pro-186 by Ala, Gly, and Val. The mutants of Pro-50 and Pro-91 all showed normal chromophore and proton pumping. However, the rates of regeneration of the chromophore in Pro-50----Ala, Pro-91----Ala and ----Gly with all-trans-retinal were about 30-fold slower than that in the wild-type, whereas the chromophore regeneration rate in Pro-50----Gly was 10-fold faster than in the wild-type. While, Pro-186----Ala regenerated the wild-type chromophore, the mutants Pro-186----Val and Pro-186----Gly showed large blue shifts (about 80 nm) in the chromophore regenerated with all-trans-retinal and showed no apparent dark-light adaptation. Pro-186----Gly first regenerated the wild-type chromophore with 13-cis-retinal which was thermally unstable and rapidly converted to the blue-shifted chromophore obtained with all-trans-retinal. High salt concentration restored the wild-type purple chromophore in the Pro-186----Gly mutant. Thus, in this mutant, the protein interconverts between two conformational states. Pro-186----Ala and Pro-186----Gly showed about 65%, whereas Pro-186----Val showed 10-20% of the normal proton pumping.     

Influence of site-directed modifications on the formation of iron cores in ferritin

The structure and crystal chemical properties of iron cores of reconstituted recombinant human ferritins and their site-directed variants have been studied by transmission electron microscopy and electron diffraction. The kinetics of Fe uptake have been compared spectrophotometrically. Recombinant L and H-chain ferritins, and recombinant H-chain variants incorporating modifications in the threefold (Asp131----His or Glu134----Ala) and fourfold (Leu169----Arg) channels, at the partially buried ferroxidase sites (Glu62,His65----Lys,Gly), a putative nucleation site on the inner surface (Glu61,Glu64,Glu67----Ala), and both the ferroxidase and nucleation sites (Glu62,His65----Lys,Gly and Glu61,Glu64,Glu67----Ala), were investigated. An additional H-chain variant, incorporating substitution of the last ten C-terminal residues for those of the L-chain protein, was also studied. Most of the proteins assimilated iron to give discrete electron-dense cores of the Fe(III) hydrated oxide, ferrihydrite (Fe2O3.nH2O). No differences were observed for variants modified in the three- or fourfold channels compared with the unmodified H-chain ferritin. The recombinant L-chain ferritin and H-chain variant depleted of the ferroxidase site, however, showed markedly reduced uptake kinetics and comprised cores of increased diameter and regularity. Depletion of the inner surface Glu residues, whilst maintaining the ferroxidase site, resulted in a partially reduced rate of Fe uptake and iron cores of wider particle size distribution. Modification of both ferroxidase and inner surface Glu residues resulted in complete inhibition of iron uptake and deposition. No cores were observed by electron microscopy although negative staining showed that the protein shell was intact. The general requirement of an appropriate spatial charge density across the cavity surface rather than specific amino acid residues could explain how, in spite of an almost complete lack of identity between the amino acid sequences of bacterioferritin and mammalian ferritins, ferrihydrite is deposited within the cavity of both proteins under similar reconstitution conditions.     

The Gly-Gly linker region of the insect cytokine growth-blocking peptide is essential for activity

Growth-blocking peptide (GBP) is a 25-amino acid cytokine isolated from the lepidopteran insect Pseudaletia separata. GBP exhibits various biological activities such as regulation of larval growth of insects, proliferation of a few kinds of cultured cells, and stimulation of a class of insect immune cells called plasmatocytes. The tertiary structure of GBP consists of a well structured core domain and disordered N and C termini. Our previous studies revealed that, in addition to the structured core, specific residues in the unstructured N-terminal region (Glu1 and Phe3) are also essential for the plasmatocyte-stimulating activity. In this study, a number of deletion, insertion, and site-directed mutants targeting the unstructured N-terminal residues of GBP were constructed to gain more detailed insight into the mode of interaction between the N-terminal region and GBP receptor. Alteration of the backbone length of the linker region between the core structure and N-terminal domain reduced plasmatocyte-stimulating activity. The substitutions of Gly5 or Gly6 in this linker region with more bulky residues, such as Phe and Pro, also remarkably reduced this activity. We conclude that the interaction of GBP with its receptor depends on the relative position of the N-terminal domain to the core structure, and therefore the backbone flexibility of Gly residues in the linker region is necessary for adoption of a proper conformation suited to receptor binding. Additionally, antagonistic experiments using deletion mutants confirmed that not only the core domain but also the N-terminal region of GBP are required for "receptor-binding," and furthermore Phe3 is a binding determinant of the N-terminal domain.     

Mutations in Ser174 and the glycine-rich sequence (Gly149, Gly150, and Thr156) in the beta subunit of Escherichia coli H(+)-ATPase.

A sequence motif in the beta subunit of Escherichia coli F1 (Gly-Gly-Ala-Gly-Val-Gly-Lys-Thr, residue 149-156, where conserved residues are underlined) is one of the glycine-rich sequences found in many nucleotide binding proteins. In this study, we constructed a plasmid carrying all the F0F1 genes. This plasmid gave the highest membrane ATPase activity so far reported. Substitution of beta Gly149 by Ser suppressed the effect of the beta Ser174----Phe mutation (defective H(+)-ATPase), but beta Gly150----Ser substitution did not have this effect. A single mutation (beta Gly149----Ser or beta Gly150----Ser) gave active enzyme with altered divalent cation dependency and azide sensitivity: the beta Gly149----Ser mutant enzyme had 100-fold lower azide sensitivity and essentially no Ca(2+)-dependent activity, but had the wild-type level of Mg(2+)-dependent activity with active oxidative phosphorylation. Introduction of a beta Gly149----Ser or beta Gly150----Ser mutation with the beta Ser174----Phe mutation also lowered the Ca(2+)-dependent activity and azide sensitivity. Consistent with our previous findings (Takeyama, M., Ihara, K., Moriyama, Y., Noumi, T., Ida, K., Tomioka, N., Itai, A., Maeda, M., and Futai, M. (1990) J. Biol. Chem. 265, 21279-21284), a beta Thr156----Ala or Cys mutation impaired ATPase activity, suggesting that the hydroxyl moiety at position 156 is essential for the catalytic activity. The possible location of the catalytic site including divalent cation binding site(s) is discussed.     

Substitution of histidine-84 and the GTPase mechanism of elongation factor Tu

Mutation of His84, a residue situated in one of the loops forming the guanine nucleotide binding pocket, was introduced in the G domain, the isolated N-terminal half molecule of bacterial elongation factor Tu (EF-Tu), in order to investigate the role of this residue on the basic activities of EF-Tu: the interaction with GDP and GTP and the hydrolysis of GTP. Substitution of His84 by Gly reduces the GTPase activity of the G domain to 5%; this activity can still be stimulated by raising the KCl concentration as the activity of wild-type G domain or the intact molecule. Since the affinities of the mutant protein for GDP and GTP are essentially the same as those of the wild-type G domain, His84 is apparently not involved in the binding of the substrates. Calculations of the change in free energy of activation of the GTPase reaction following substitution of His84 by Gly point to the disruption of a weak hydrogen bond, involved in the catalytic reaction. This probably concerns an interaction via a water molecule. The possible mechanism underlying the GTPase reaction is discussed in light of the three-dimensional structure of EF-Tu, taking into account the situation of Ha-ras p21.     

Characterization of a self-splicing mini-intein and its conversion into autocatalytic N- and C-terminal cleavage elements: facile production of protein building blocks for protein ligation

The determinants governing the self-catalyzed splicing and cleavage events by a mini-intein of 154 amino acids, derived from the dnaB gene of Synechocystis sp. were investigated. The residues at the splice junctions have a profound effect on splicing and peptide bond cleavage at either the N- or C-terminus of the intein. Mutation of the native Gly residue preceding the intein blocked splicing and cleavage at the N-terminal splice junction, while substitution of the intein C-terminal Asn154 resulted in the modulation of N-terminal cleavage activity. Controlled cleavage at the C-terminal splice junction involving cyclization of Asn154 was achieved by substitution of the intein N-terminal cysteine residue with alanine and mutation of the native C-extein residues. The C-terminal cleavage reaction was found to be pH-dependent, with an optimum between pH6.0 and 7.5. These findings allowed the development of single junction cleavage vectors for the facile production of proteins as well as protein building blocks with complementary reactive groups. A protein sequence was fused to either the N-terminus or C-terminus of the intein, which was fused to a chitin binding domain. The N-terminal cleavage reaction was induced by 2-mercaptoethanesulfonic acid and released the 43kDa maltose binding protein with an active C-terminal thioester. The 58kDa T4 DNA ligase possessing an N-terminal cysteine was generated by a C-terminal cleavage reaction induced by pH and temperature shifts. The intein-generated proteins were joined together through a native peptide bond. This intein-mediated protein ligation approach opens up novel routes in protein engineering.     

Selective cell-surface expression of dipeptidyl peptidase IV with mutations at the active site sequence.

The cell surface expression of dipeptidyl peptidase IV (DPPIV) was examined in COS-1 cells transfected with its cDNA with or without mutations at the active site sequence Gly-Trp-Ser-Tyr-Gly (positions 629-633). Mutants with substitution of Trp630----Glu or Ser631----Ala were expressed on the cell surface as normally as the wild-type DPPIV, although the mutant with Ala631 had no enzyme activity. In contrast, any single substitutions of Gly at positions 629 and 633 resulted in no surface expression of the mutants, which were, instead, detected within the cells. When Tyr632 was substituted, one mutant (Tyr----Phe) was expressed on the surface, whereas the others (Tyr----Gly or Leu) were intracellularly retained. These results indicate that the surface expression of DPPIV is critically influenced by mutations at the active site sequence.     

Mutational analysis and membrane-interactions of the beta-sheet-like N-terminal domain of the pediocin-like antimicrobial peptide sakacin P

To gain insight into how the N-terminal three-stranded beta-sheet-like domain in pediocin-like antimicrobial peptides positions itself on membranes, residues in the well-conserved (Y)YGNGV-motif in the domain were substituted and the effect of the substitutions on antimicrobial activity and binding of peptides to liposomes was determined. Peptide-liposome interactions were detected by measuring tryptophan-fluorescence upon exposing liposomes to peptides in which a tryptophan residue had been introduced in the N-terminal domain. The results revealed that the N-terminal domain associates readily with anionic liposomes, but not with neutral liposomes. The electrostatic interactions between peptides and liposomes facilitated the penetration of some of the peptide residues into the liposomes. Measuring the antimicrobial activity of the mutated peptides revealed that the Tyr2Leu and Tyr3Leu mutations resulted in about a 10-fold reduction in activity, whereas the Tyr2Trp, Tyr2Phe, Tyr3Trp and Tyr3Phe mutations were tolerated fairly well, especially the mutations in position 3. The Val7Ile mutation did not have a marked detrimental effect on the activity. The Gly6Ala mutation was highly detrimental, consistent with Gly6 being in one of the turns in the beta-sheet-like N-terminal domain, whereas the Gly4Ala mutation was tolerated fairly well. All mutations involving Asn5, including the conservative mutations Asn5Gln and Asn5Asp, were very deleterious. Thus, both the polar amide group on the side chain of Asn5 and its exact position in space were crucial for the peptides to be fully active. Taken together, the results are consistent with Val7 positioning itself in the hydrophobic core of target membranes, thus forcing most of the other residues in the N-terminal domain into the membrane interface region: Tyr3 and Asn5 in the lower half with their side chains pointing downward and approaching the hydrophobic core, Tyr2, Gly4 and His8 and 12 in the upper half, Lys1 near the middle of the interface region, and the side chain of Lys11 pointing out toward the membrane surface.     

Analysis of the substrate specificity loop of the HAD superfamily cap domain

The haloacid dehalogenase (HAD) superfamily includes a variety of enzymes that catalyze the cleavage of substrate C-Cl, P-C, and P-OP bonds via nucleophilic substitution pathways. All members possess the alpha/beta core domain, and many also possess a small cap domain. The active site of the core domain is formed by four loops (corresponding to sequence motifs 1-4), which position substrate and cofactor-binding residues as well as the catalytic groups that mediate the "core" chemistry. The cap domain is responsible for the diversification of chemistry within the family. A tight beta-turn in the helix-loop-helix motif of the cap domain contains a stringently conserved Gly (within sequence motif 5), flanked by residues whose side chains contribute to the catalytic site formed at the domain-domain interface. To define the role of the conserved Gly in the structure and function of the cap domain loop of the HAD superfamily members phosphonoacetaldehyde hydrolase and beta-phosphoglucomutase, the Gly was mutated to Pro, Val, or Ala. The catalytic activity was severely reduced in each mutant. To examine the impact of Gly substitution on loop 5 conformation, the X-ray crystal structure of the Gly50Pro phosphonoacetaldehyde hydrolase mutant was determined. The altered backbone conformation at position 50 had a dramatic effect on the spatial disposition of the side chains of neighboring residues. Lys53, the Schiff Base forming lysine, had rotated out of the catalytic site and the side chain of Leu52 had moved to fill its place. On the basis of these studies, it was concluded that the flexibility afforded by the conserved Gly is critical to the function of loop 5 and that it is a marker by which the cap domain substrate specificity loop can be identified within the amino acid sequence of HAD family members.     

Osteogenesis Imperfecta Model Peptides: Incorporation of Residues Replacing Gly within a Triple Helix Achieved by Renucleation and Local Flexibility

Missense mutations, which replace one Gly with a larger residue in the repeating sequence of the type I collagen triple helix, lead to the hereditary bone disorder osteogenesis imperfecta (OI). Previous studies suggest that these mutations may interfere with triple-helix folding. NMR was used to investigate triple-helix formation in a series of model peptides where the residue replacing Gly, as well as the local sequence environment, was varied. NMR measurement of translational diffusion coefficients allowed the identification of partially folded species. When Gly was replaced by Ala, the Ala residue was incorporated into a fully folded triple helix, whereas replacement of Gly by Ser or Arg resulted in the presence of some partially folded species, suggesting a folding barrier. Increasing the triple-helix stability of the sequence N-terminal to a Gly-to-Ser replacement allowed complete triple-helix folding, whereas with the substitution of Arg, with its large side chain, the peptide achieved full folding only after flexible residues were introduced N-terminal to the mutation site. These studies shed light on the factors important for accommodation of Gly mutations within the triple helix and may relate to the varying severity of OI.     

Functional consequences of mutations of conserved amino acids in the beta-strand domain of the Ca2(+)-ATPase of sarcoplasmic reticulum

The sequences Thr-Gly-Glu-Ser184 and Asp-Gln-Ser178 and individual residues Asp149, Asp157, and Asp162 in the sarcoplasmic reticulum Ca2(+)-ATPase are highly conserved throughout the family of cation-transporting ATPases. Mutant Thr181----Ala, Gly182----Ala, Glu183----Ala, and Glu183----Gln, created by in vitro mutagenesis, were devoid of Ca2+ transport activity. None of these mutations, however, affected phosphorylation of the enzyme by ATP in the presence of Ca2+ or by inorganic phosphate in the absence of Ca2+, indicating that the high affinity Ca2(+)-binding sites and the nucleotide-binding sites were intact. In each of these mutants, the ADP-sensitive phosphoenzyme intermediate (E1P) decayed to the ADP-insensitive form (E2P) very slowly relative to the wild-type enzyme, whereas E2P decayed at a rate similar to that of the wild-type enzyme. Thus, the inability of the mutants to transport Ca2+ was accounted for by an apparent block of the transport reaction at the E1P to E2P conformational transition. These results suggest that Thr181, Gly182, and Glu183 play essential roles in the conformational change between E1P and E2P. Mutation of Ser184, Asp157, or Ser178 had little or no effect on either Ca2+ transport activity or expression. Mutations of Asp149, Asp162, and Gln177, however, were poorly expressed. Where expression could be measured, in mutations to Asp162 and Gln177, Ca2+ transport activity was essentially equivalent to that of the wild-type enzyme.     

Essential, completely conserved glycine residue in the domain III S2-S3 linker of voltage-gated calcium channel alpha1 subunits in yeast and mammals

Voltage-gated Ca2+ channels (VGCCs) mediate the influx of Ca2+ that regulates many cellular events, and mutations in VGCC genes cause serious hereditary diseases in mammals. The yeast Saccharomyces cerevisiae has only one gene encoding the putative pore-forming alpha1 subunit of VGCC, CCH1. Here, we identify a cch1 allele producing a completely nonfunctional Cch1 protein with a Gly1265 to Glu substitution present in the domain III S2-S3 cytoplasmic linker. Comparison of amino acid sequences of this linker among 58 VGCC alpha1 subunits from 17 species reveals that a Gly residue whose position corresponds to that of the Cch1 Gly1265 is completely conserved from yeasts to humans. Systematic amino acid substitution analysis using 10 amino acids with different chemical and structural properties indicates that the Gly1265 is essential for Cch1 function because of the smallest residue volume. Replacement of the Gly959 residue of a rat brain Cav1.2 alpha1 subunit (rbCII), positionally corresponding to the yeast Cch1 Gly1265, with Glu, Ser, Lys, or Ala results in the loss of Ba2+ currents, as revealed by the patch clamp method. These results suggest that the Gly residue in the domain III S2-S3 linker is functionally indispensable from yeasts to mammals. Because the Gly residue has never been studied in any VGCC, these findings provide new insights into the structure-function relationships of VGCCs.