The amino acid sequence of human chorionic gonadotropin. The alpha subunit and beta subunit.

The amino acid sequences of both the alpha and beta subunits of human chorionic gonadotropin have been determined. The amino acid sequence of the alpha subunit is: Ala - Asp - Val - Gln - Asp - Cys - Pro - Glu - Cys-10 - Thr - Leu - Gln - Asp - Pro - Phe - Ser - Gln-20 - Pro - Gly - Ala - Pro - Ile - Leu - Gln - Cys - Met - Gly-30 - Cys - Cys - Phe - Ser - Arg - Ala - Tyr - Pro - Thr - Pro-40 - Leu - Arg - Ser - Lys - Lys - Thr - Met - Leu - Val - Gln-50 - Lys - Asn - Val - Thr - Ser - Glu - Ser - Thr - Cys - Cys-60 - Val - Ala - Lys - Ser - Thr - Asn - Arg - Val - Thr - Val-70 - Met - Gly - Gly - Phe - Lys - Val - Glu - Asn - His - Thr-80 - Ala - Cys - His - Cys - Ser - Thr - Cys - Tyr - Tyr - His-90 - Lys - Ser. Oligosaccharide side chains are attached at residues 52 and 78. In the preparations studied approximately 10 and 30% of the chains lack the initial 2 and 3 NH2-terminal residues, respectively. This sequence is almost identical with that of human luteinizing hormone (Sairam, M. R., Papkoff, H., and Li, C. H. (1972) Biochem. Biophys. Res. Commun. 48, 530-537). The amino acid sequence of the beta subunit is: Ser - Lys - Glu - Pro - Leu - Arg - Pro - Arg - Cys - Arg-10 - Pro - Ile - Asn - Ala - Thr - Leu - Ala - Val - Glu - Lys-20 - Glu - Gly - Cys - Pro - Val - Cys - Ile - Thr - Val - Asn-30 - Thr - Thr - Ile - Cys - Ala - Gly - Tyr - Cys - Pro - Thr-40 - Met - Thr - Arg - Val - Leu - Gln - Gly - Val - Leu - Pro-50 - Ala - Leu - Pro - Gin - Val - Val - Cys - Asn - Tyr - Arg-60 - Asp - Val - Arg - Phe - Glu - Ser - Ile - Arg - Leu - Pro-70 - Gly - Cys - Pro - Arg - Gly - Val - Asn - Pro - Val - Val-80 - Ser - Tyr - Ala - Val - Ala - Leu - Ser - Cys - Gln - Cys-90 - Ala - Leu - Cys - Arg - Arg - Ser - Thr - Thr - Asp - Cys-100 - Gly - Gly - Pro - Lys - Asp - His - Pro - Leu - Thr - Cys-110 - Asp - Asp - Pro - Arg - Phe - Gln - Asp - Ser - Ser - Ser - Ser - Lys - Ala - Pro - Pro - Pro - Ser - Leu - Pro - Ser-130 - Pro - Ser - Arg - Leu - Pro - Gly - Pro - Ser - Asp - Thr-140 - Pro - Ile - Leu - Pro - Gln. Oligosaccharide side chains are found at residues 13, 30, 121, 127, 132, and 138. The proteolytic enzyme, thrombin, which appears to cleave a limited number of arginyl bonds, proved helpful in the determination of the beta sequence.     

Crystal structure of a highly acidic neurotoxin from scorpion Buthus tamulus at 2.2A resolution reveals novel structural features

The crystal structure of a highly acidic neurotoxin from the scorpion Buthus tamulus has been determined at 2.2A resolution. The amino acid sequence determination shows that the polypeptide chain has 64 amino acid residues. The pI measurement gave a value of 4.3 which is one of the lowest pI values reported so far for a scorpion toxin. As observed in other alpha-toxins, it contains four disulphide bridges, Cys12-Cys63, Cys16-Cys36, Cys22-Cys46, and Cys26-Cys48. The crystal structure reveals the presence of two crystallographically independent molecules in the asymmetric unit. The conformations of two molecules are identical with an r.m.s. value of 0.3A for their C(alpha) tracings. The overall fold of the toxin is very similar to other scorpion alpha-toxins. It is a betaalphabetabeta protein. The beta-sheet involves residues Glu2-Ile6 (strand beta1), Asp32-Trp39 (strand beta3) and Val45-Val55 (strand beta4). The single alpha-helix formed is by residues Asn19-Asp28 (alpha2). The structure shows a trans peptide bond between residues 9 and 10 in the five-membered reverse turn Asp8-Cys12. This suggests that this toxin belongs to classical alpha-toxin subfamily. The surface features of the present toxin are highly characteristic, the first (A-site) has residues, Phe18, Trp38 and Trp39 that protrude outwardly presumably to interact with its receptor. There is another novel face (N-site) of this neurotoxin that contains several negatively charged residues such as, Glu2, Asp3, Asp32, Glu49 and Asp50 which are clustered in a small region of the toxin structure. On yet another face (P-site) in a triangular arrangement, with respect to the above two faces there are several positively charged residues, Arg58, Lys62 and Arg64 that also protrude outwardly for a potentially potent interaction with other molecules. This toxin with three strong features appears to be one of the most toxic molecules reported so far. In this sense, it may be a new subclass of neurotoxins with the largest number of hot spots.     

Role of the invariant aspartic acid 99 of human choriogonadotropin beta in receptor binding and biological activity

The four human glycoprotein hormones are heterodimers that contain a common alpha subunit and a hormone-specific beta subunit. Within this hormone family, 23 amino acid sequences from 11 mammalian species are available. There are 19 invariant amino acid residues in the beta subunits, 12 of which are Cys that form six disulfide bonds. Of the remaining seven conserved amino acid residues, we have investigated the role of an Asp which occurs at position 99 in human choriogonadotropin beta (hCG beta). Site-directed mutagenesis was used to replace hCG beta Asp99 with three residues, Glu, Asn, and Arg, and to prepare an inversion double mutant protein, Arg94----Asp and Asp99----Arg. The cDNAs were placed in a eukaryotic expression vector, and the plasmids were transiently transfected into Chinese hamster ovary cells containing a stably integrated gene for bovine alpha. Radioimmunoassays demonstrated that the mutant forms of hCG beta were capable of subunit assembly to the same extent as hCG beta wild type. The heterologous heterodimers were assayed in vitro using transformed mouse Leydig cells (MA-10) by competitive inhibition of 125I-hCG binding and stimulation of progesterone production. The gonadotropins containing Glu and Asn were active, although the potency was less than that associated with the hCG beta wild type-containing gonadotropin. In contrast, the Arg99-containing mutant protein and the inversion mutant protein Asp94/Arg99 were devoid of activity. Thus, in hCG beta Asp99 can be substituted with certain residues without total loss of function, although replacement with a positively charged residue leads to an inactive heterodimer. The primary role of Asp99 in hCG beta seems to involve, either directly or indirectly, receptor recognition.     

Dissection of the energetic coupling across the Src SH2 domain-tyrosyl phosphopeptide interface

Src Homology (SH2) domains play critical roles in signaling pathways by binding to phosphotyrosine (pTyr)-containing sequences, thereby recruiting SH2 domain-containing proteins to tyrosine-phosphorylated sites on receptor molecules. Investigations of the peptide binding specificity of the SH2 domain of the Src kinase (Src SH2 domain) have defined the EEI motif C-terminal to the phosphotyrosine as the preferential binding sequence. A subsequent study that probed the importance of eight specificity-determining residues of the Src SH2 domain found two residues which when mutated to Ala had significant effects on binding: Tyr beta D5 and Lys beta D3. The mutation of Lys beta D3 to Ala was particularly intriguing, since a Glu to Ala mutation at the first (+1) position of the EEI motif (the residue interacting with Lys beta D3) did not significantly affect binding. Hence, the interaction between Lys beta D3 and +1 Glu is energetically coupled. This study is focused on the dissection of the energetic coupling observed across the SH2 domain-phosphopeptide interface at and around the +1 position of the peptide. It was found that three residues of the SH2 domain, Lys beta D3, Asp beta C8 and AspCD2 (altogether forming the so-called +1 binding region) contribute to the selection of Glu at the +1 position of the ligand. A double (Asp beta C8Ala, AspCD2Ala) mutant does not exhibit energetic coupling between Lys beta D3 and +1 Glu, and binds to the pYEEI sequence 0.3 kcal/mol tighter than the wild-type Src SH2 domain. These results suggest that Lys beta D3 in the double mutant is now free to interact with the +1 Glu and that the role of Lys beta D3 in the wild-type is to neutralize the acidic patch formed by Asp beta C8 and AspCD2 rather than specifically select for a Glu at the +1 position as it had been hypothesized previously. A triple mutant (Lys beta D3Ala, Asp beta C8Ala, AspCD2Ala) has reduced binding affinity compared to the double (Asp beta C8Ala, AspCD2Ala) mutant, yet binds the pYEEI peptide as well as the wild-type Src SH2 domain. The structural basis for such high affinity interaction was investigated crystallographically by determining the structure of the triple (Lys beta D3Ala, Asp beta C8Ala, AspCD2Ala) mutant bound to the octapeptide PQpYEEIPI (where pY indicates a phosphotyrosine). This structure reveals for the first time contacts between the SH2 domain and the -1 and -2 positions of the peptide (i.e. the two residues N-terminal to pY). Thus, unexpectedly, mutations in the +1 binding region affect binding of other regions of the peptide. Such additional contacts may account for the high affinity interaction of the triple mutant for the pYEEI-containing peptide.     

Amino acid residue penultimate to the amino-terminal gly residue strongly affects two cotranslational protein modifications, N-myristoylation and N-acetylation

To examine the amino-terminal sequence requirements for cotranslational protein N-myristoylation, a series of site-directed mutagenesis of N-terminal region were performed using tumor necrosis factor as a nonmyristoylated model protein. Subsequently, the susceptibility of these mutants to protein N-myristoylation was evaluated by metabolic labeling in an in vitro translation system or in transfected cells. It was found that the amino acid residue at position 3 in an N-myristoylation consensus motif, Met-Gly-X-X-X-Ser-X-X-X, strongly affected the susceptibility of the protein to two different cotranslational protein modifications, N-myristoylation and N-acetylation; 10 amino acids (Ala, Ser, Cys, Thr, Val, Asn, Leu, Ile, Gln, and His) with a radius of gyration smaller than 1.80 A directed N-myristoylation, two negatively charged residues (Asp and Glu) directed N-acetylation, and two amino acids (Gly and Met) directed heterogeneous modification with both N-myristoylation and N-acetylation. The amino acid requirements at this position for the two modifications were dramatically changed when Ser at position 6 in the consensus motif was replaced with Ala. Thus, the amino acid residue penultimate to the N-terminal Gly residue strongly affected two cotranslational protein modifications, N-myristoylation and N-acetylation, and the amino acid requirements at this position for these two modifications were significantly affected by downstream residues.     

Identification by mutational analysis of amino acid residues essential in the chaperone function of calreticulin

Calreticulin is a Ca2+ -binding chaperone that resides in the lumen of the endoplasmic reticulum and is involved in the regulation of intracellular Ca2+ homeostasis and in the folding of newly synthesized glycoproteins. In this study, we have used site-specific mutagenesis to map amino acid residues that are critical in calreticulin function. We have focused on two cysteine residues (Cys(88) and Cys(120)), which form a disulfide bridge in the N-terminal domain of calreticulin, on a tryptophan residue located in the carbohydrate binding site (Trp(302)), and on certain residues located at the tip of the "hairpin-like" P-domain of the protein (Glu(238), Glu(239), Asp(241), Glu(243), and Trp(244)). Calreticulin mutants were expressed in crt(-/-) fibroblasts, and bradykinin-dependent Ca2+ release was measured as a marker of calreticulin function. Bradykinin-dependent Ca2+ release from the endoplasmic reticulum was rescued by wild-type calreticulin and by the Glu(238), Glu(239), Asp(241), and Glu(243) mutants. The Cys(88) and Cys(120) mutants rescued the calreticulin-deficient phenotype only partially ( approximately 40%), and the Trp(244) and Trp(302) mutants did not rescue it at all. We identified four amino acid residues (Glu(239), Asp(241), Glu(243), and Trp(244)) at the hairpin tip of the P-domain that are critical in the formation of a complex between ERp57 and calreticulin. Although the Glu(239), Asp(241), and Glu(243) mutants did not bind ERp57 efficiently, they fully restored bradykinin-dependent Ca2+ release in crt(-/-) cells. This indicates that binding of ERp57 to calreticulin may not be critical for the chaperone function of calreticulin with respect to the bradykinin receptor.     

Role of acidic residues as substrate determinants for casein kinase I

Sites phosphorylated by casein kinase I have been characterized by the presence of acidic amino acids NH2-terminal to the modified residue. Recently, phosphoserine was shown to be a particularly effective determinant for casein kinase I action when present in the motif -S(P)-X-X-S- (Flotow, H., Graves, P. R., Wang, A., Fiol, C. J., Roeske, R. W., and Roach, P. J. (1990) J. Biol. Chem. 265, 14264-14269). Nonetheless, nonphosphorylated substrates for casein kinase I are well documented. In this study, we examined the efficacy of Asp and Glu residues as determinants of casein kinase I action using synthetic peptide substrates. Peptides with runs of Asp residues in the motif Dn-X-X-S- were substrates for casein kinase I. Peptides with n = 3 or 4 were the most effective substrates, much better than n = 2. The peptide with n = 1, a single Asp residue, was a very poor substrate. A block of 4 Glu residues was a little less effective as a substrate determinant than 4 Asp residues in an otherwise identical peptide. The most effective substrate, with the motif -D-D-D-D-X-X-S-, was specific for casein kinase I and was not detectably phosphorylated by cyclic AMP-dependent protein kinase, casein kinase II, glycogen synthase kinase 3, or phosphorylase kinase and thus will be useful for the specific assay of casein kinase I. This peptide was nonetheless significantly worse as a substrate than peptides in which casein kinase I action was determined by phosphoserine in the -3 position. Still, the fact that Asp or Glu residues can specify a casein kinase I substrate suggests that acidic character has a role in substrate selection by this protein kinase.     

Chicken mitochondrial cytochrome C oxidase subunit II: comparative analysis among the vertebrates

The chicken cytochrome c oxidase subunit II (COII) was cloned and sequenced. A comparison of the deduced chicken COII sequence with 4 other vertebrate counterparts revealed 64-66% amino acid sequence homology and 68-70% nucleotide sequence homology. Four peptide segments each of nine amino acids long are highly conserved across the 5 species. A redox-center was formed by three of these highly conserved domains, which include two invariant Cys and two invariant His residues for copper ion coordination, three strictly conserved Glu or Asp residues for cytochrome c binding, and highly conserved aromatic acid residues for electron transfer.     

Ligand binding properties and structural studies of recombinant and chemically modified hemoglobins altered at beta 93 cysteine

To investigate the roles of beta93 cysteine in human normal adult hemoglobin (Hb A), we have constructed four recombinant mutant hemoglobins (rHbs), rHb (betaC93G), rHb (betaC93A), rHb (betaC93M), and rHb (betaC93L), and have prepared two chemically modified Hb As, Hb A-IAA and Hb A-NEM, in which the sulfhydryl group at beta93Cys is modified by sulfhydryl reagents, iodoacetamide (IAA) and N-ethylmaleimide (NEM), respectively. These variants at the beta93 position show higher oxygen affinity, lower cooperativity, and reduced Bohr effect relative to Hb A. The response of some of these Hb variants to allosteric effectors, 2,3-bisphosphoglycerate (2,3-BPG) and inositol hexaphosphate (IHP), is decreased relative to that of Hb A. The proton nuclear magnetic resonance (NMR) spectra of these Hb variants show that there is a marked influence on the proximal heme pocket of the beta-chain, whereas the environment of the proximal heme pocket of the alpha-chain remains unchanged as compared to Hb A, suggesting that higher oxygen affinity is likely to be determined by the heme pocket of the beta-chain rather than by that of the alpha-chain. This is further supported by NO titration of these Hbs in the deoxy form. For Hb A, NO binds preferentially to the heme of the alpha-chain relative to that of the beta-chain. In contrast, the feature of preferential binding to the heme of the alpha-chain becomes weaker and even disappears for Hb variants with modifications at beta93Cys. The effects of IHP on these Hbs in the NO form are different from those on HbNO A, as characterized by (1)H NMR spectra of the T-state markers, the exchangeable resonances at 14 and 11 ppm, reflecting that these Hb variants have more stability in the R-state relative to Hb A, especially rHb (betaC93L) and Hb A-NEM in the NO form. The changes of the C2 proton resonances of the surface histidyl residues in these Hb variants in both the deoxy and CO forms, compared with those of Hb A, indicate that a mutation or chemical modification at beta93Cys can result in conformational changes involving several surface histidyl residues, e.g., beta146His and beta2His. The results obtained here offer strong evidence to show that the salt bridge between beta146His and beta94Asp and the binding pocket of allosteric effectors can be affected as the result of modifications at beta93Cys, which result in the destabilization of the T-state and a reduced response of these Hbs to allosteric effectors. We further propose that the impaired alkaline Bohr effect can be attributed to the effect on the contributions of several surface histidyl residues which are altered because of the environmental changes caused by mutations and chemical modifications at beta93Cys.     

Mercurial activation of human PMN leucocyte type IV procollagenase (gelatinase).

Autoproteolytic activation and processing of human polymorphonuclear leucocyte (PMNL) type IV procollagenase (gelatinase) was initiated by HgCl2 and was investigated by kinetic analysis and N-terminal sequence determination of the reaction products. In the first instance the propeptide domain was lost by subsequent cleavage of the Asp15-Leu16, Glu40-Met41, Leu52-Leu53 and Ala74-Met75 peptide bonds. The PRCGVPD sequence motif (residues Pro78-Asp84), which is conserved in all metalloproteinases and expected to be relevant for latency, remained uncleaved at the activated enzyme. The generated intermediate was further processed by three C-terminal cleavages. The Glu666-Leu667, Ala506-Glu507 and Ala398-Leu399 bonds were hydrolysed successively. From the fragmentation products we were able to conclude that three released fragment peptides contained unpaired free cysteine with the residues Cys497, Cys653, Cys683. Cleavage of the first C-terminal peptide bond resulted in the loss of one of the conserved Cys residues of the hemopexin-like domain, whereas the Cys residue of the PRCGVPD motif was retained at the fully active enzyme. The possibility of an entirely different activation mechanism for PMNL type IV procollagenase is discussed.     

Identification of a novel conserved sequence motif in flavoprotein hydroxylases with a putative dual function in FAD/NAD(P)H binding.

A novel conserved sequence motif has been located among the flavoprotein hydroxylases. Based on the crystal structure and site-directed mutagenesis studies of p-hydroxybenzoate hydroxylase (PHBH) from Pseudomonas fluorescens, this amino acid fingerprint sequence is proposed to play a dual function in both FAD and NAD(P)H binding. In PHBH, the novel sequence motif (residues 153-166) includes strand A4 and the N-terminal part of helix H7. The conserved amino acids Asp 159, Gly 160, and Arg 166 are necessary for maintaining the structure. The backbone oxygen of Cys 158 and backbone nitrogens of Gly 160 and Phe 161 interact indirectly with the pyrophosphate moiety of FAD, whereas it is known from mutagenesis studies that the side chain of the moderately conserved His 162 is involved in NADPH binding.     

Agonist binding site of Torpedo electric tissue nicotinic acetylcholine receptor. A negatively charged region of the delta subunit within 0.9 nm of the alpha subunit binding site disulfide

The positively charged quaternary ammonium group of agonists of the nicotinic acetylcholine (ACh) receptor binds to a negative subsite at most about 1 nm from a readily reducible disulfide. This disulfide is formed by alpha Cys192 and Cys193 (Kao and Karlin, 1986). In order to identify Asp or Glu residues that may contribute to the negative subsite, we synthesized S-(2-[3H]glycylamidoethyl)dithio-2-pyridine. Purified ACh receptor from Torpedo californica was mildly reduced and reacted with S-(2-[3H]glycylamidoethyl)dithio-2-pyridine. The predominant product was a mixed disulfide between the 3H-N-glycylcysteamine moiety and alpha Cys192 or Cys193. In the extended conformation of [3H] N-glycylcysteamine, the distance from the glycyl amino group to the cysteamine thio group is 0.9 nm. Thus, the amino group of disulfide-linked [3H]N-glycylcysteamine could react with carboxyls within 0.9 nm of Cys192/Cys193. To promote amide bond formation between the tethered amino group and receptor carboxyls, we added 1-ethyl-3-(3'-dimethylaminopropyl)-carbodiimide. The predominant sites of amide coupling were on the delta subunit, in CNBr fragment 4 (delta 164-257). This reaction was inhibited by ACh. Only the first 61 residues of delta CNBr 4 are predicted to be extracellular, and there are 11 Asp or Gly residues in this region. One or more of these residues is likely to contribute to the binding of ACh.     

Amino acid sequence of a protease inhibitor isolated from Sarcophaga bullata determined by mass spectrometry.

The amino acid sequence of a protease inhibitor isolated from the hemolymph of Sarcophaga bullata larvae was determined by tandem mass spectrometry. Homology considerations with respect to other protease inhibitors with known primary structures assisted in the choice of the procedure followed in the sequence determination and in the alignment of the various peptides obtained from specific chemical cleavage at cysteines and enzyme digests of the S. bullata protease inhibitor. The resulting sequence of 57 residues is as follows: Val Asp Lys Ser Ala Cys Leu Gln Pro Lys Glu Val Gly Pro Cys Arg Lys Ser Asp Phe Val Phe Phe Tyr Asn Ala Asp Thr Lys Ala Cys Glu Glu Phe Leu Tyr Gly Gly Cys Arg Gly Asn Asp Asn Arg Phe Asn Thr Lys Glu Glu Cys Glu Lys Leu Cys Leu.     

The atomic-resolution structure of a novel bacterial esterase

Background: A novel bacterial esterase that cleaves esters on halogenated cyclic compounds has been isolated from an Alcaligenes species. This esterase 713 is encoded by a 1062 base pair gene. The presence of a leader sequence of 27 amino acids suggests that this enzyme is exported from the cytosol. Esterase 713 has been over-expressed in Agrobacterium without this leader sequence. Its amino acid sequence shows no significant homology to any known protein sequence. Results: The crystal structure of esterase 713 has been determined by multiple isomorphous replacement and refined to 1. 1 A resolution. The subunits of this dimeric enzyme comprise a single domain with an alpha/beta hydrolase fold. The catalytic triad has been identified as Ser206-His298-Glu230. The acidic residue of the catalytic triad (Glu230) is located on the beta6 strand of the alpha/beta hydrolase fold, whereas most other alpha/beta hydrolase enzymes have the acidic residue located on the beta7 strand. The oxyanion hole is formed by the mainchain nitrogens of Cys71 and Gln207 as identified by the binding of a substrate analogue, (S)-7-iodo-2,3,4,5-tetrahydro-4-methyl-3-oxo-1H-1, 4-benzodiazepine-2-acetic acid. Cys71 forms a disulphide bond with the neighbouring Cys72. Conclusions: Despite negligible sequence homology, esterase 713 has structural similarities to a number of other esterases and lipases. Residues of the oxyanion hole were confirmed by structural comparison with Rhizomucor miehei lipase. It is proposed that completion of a functional active site requires the formation of the disulphide bond between adjacent residues Cys71 and Cys72 on export of the esterase into the oxidising environment of the periplasmic space.     

Minimum length of an idiotypic peptide and a model for its binding to a major histocompatibility complex class II molecule.

We have defined the minimum length of a synthetic peptide which can activate I-Ed-restricted BALB/c T cell clones specific for a mutated self-antigen: an idiotope on the syngeneic lambda 2315 immunoglobulin light chain. A peptide comprising residues 91-101 of the lambda 2315 sequence had full stimulatory potency. Surprisingly, a peptide analogue in which His97 was deleted was almost fully active. Truncated, deleted or substituted peptide analogues did not distinguish between seven T cell clones that use different alpha/beta T cell receptors. The 91-101 region in the lambda 2315 light chain does not form an amphipathic helix even though such a helix has been suggested to be important for T cell epitopes. Further, a motif proposed by Rothbard and Taylor as being common to T cell immunogenic peptides is not necessary for the lambda 2315 idiotypic peptide. Comparison with seven other I-Ed-restricted peptides revealed that the peptides are generally positively charged and have two basic amino acids clustered around the centre. On the basis of a model of the class II molecule peptide binding site, we suggest that these positively charged residues may interact with the negatively charged residues at positions 114(Glu) and 155(Asp) of the E beta d chain.     

Transport of taurocholate by mutants of negatively charged amino acids, cysteines, and threonines of the rat liver sodium-dependent taurocholate cotransporting polypeptide Ntcp.

The relevance of functional amino acids for taurocholate transport by the sodium-dependent taurocholate cotransporting polypeptide Ntcp was determined by site-directed mutagenesis. cRNA from 28 single-points mutants of the rat liver Ntcp clone was expressed in Xenopus laevis oocytes. Mutations were generated in five conserved negatively charged amino acids (aspartates and glutamates) which were present in nine members of the SBAT-family, in two nonconserved negatively charged amino acids, in all eight Ntcp-cysteines, and in two threonines from a protein kinase C consensus region of the Ntcp C-terminus. Functional amino acids were Asp115, Glu257, and Cys266, which were found to be essential for the maintenance of taurocholic acid transport. Asp115 is located in the large intracellular loop III, whereas Glu257 and Cys266 are located in the large extracellular loop VI. Four mutations of threonines from the C-terminus of the Ntcp by alanines or tyrosines showed no effects on sodium-dependent taurocholate transport. Introduction of the FLAG(R) motif into several transport negative point mutations demonstrated that all mutated proteins besides one were present within the cell membrane of the oocytes and provided proof that an insertion defect has not caused transport deficiency by these Ntcp mutants. The latter was observed only with the transport negative mutant Asp24Asn. In conclusion, loop amino acids are required for sodium-dependent substrate translocation by the Ntcp.     

Identification of structural and catalytic classes of highly conserved amino acid residues in lysine 2,3-aminomutase

Lysine 2,3-aminomutase (LAM) from Clostridium subterminale SB4 catalyzes the interconversion of (S)-lysine and (S)-beta-lysine by a radical mechanism involving coenzymatic actions of S-adenosylmethionine (SAM), a [4Fe-4S] cluster, and pyridoxal 5'-phosphate (PLP). The enzyme contains a number of conserved acidic residues and a cysteine- and arginine-rich motif, which binds iron and sulfide in the [4Fe-4S] cluster. The results of activity and iron, sulfide, and PLP analysis of variants resulting from site-specific mutations of the conserved acidic residues and the arginine residues in the iron-sulfide binding motif indicate two classes of conserved residues of each type. Mutation of the conserved residues Arg134, Asp293, and Asp330 abolishes all enzymatic activity. On the basis of the X-ray crystal structure, these residues bind the epsilon-aminium and alpha-carboxylate groups of (S)-lysine. However, among these residues, only Asp293 appears to be important for stabilizing the [4Fe-4S] cluster. Members of a second group of conserved residues appear to stabilize the structure of LAM. Mutations of arginine 130, 135, and 136 and acidic residues Glu86, Asp165, Glu236, and Asp172 dramatically decrease iron and sulfide contents in the purified variants. Mutation of Asp96 significantly decreases iron and sulfide content. Arg130 or Asp172 variants display no detectable activity, whereas variants mutated at the other positions display low to very low activities. Structural roles are assigned to this latter class of conserved amino acids. In particular, a network of hydrogen bonded interactions of Arg130, Glu86, Arg135, and the main chain carbonyl groups of Cys132 and Leu55 appears to stabilize the [4Fe-4S] cluster.     

Heparan/chondroitin sulfate biosynthesis. Structure and mechanism of human glucuronyltransferase I

Human beta1,3-glucuronyltransferase I (GlcAT-I) is a central enzyme in the initial steps of proteoglycan synthesis. GlcAT-I transfers a glucuronic acid moiety from the uridine diphosphate-glucuronic acid (UDP-GlcUA) to the common linkage region trisaccharide Gal beta 1-3Gal beta 1-4Xyl covalently bound to a Ser residue at the glycosaminylglycan attachment site of proteoglycans. We have now determined the crystal structure of GlcAT-1 at 2.3 A in the presence of the donor substrate product UDP, the catalytic Mn(2+) ion, and the acceptor substrate analog Gal beta 1-3Gal beta 1-4Xyl. The enzyme is a alpha/beta protein with two subdomains that constitute the donor and acceptor substrate binding site. The active site residues lie in a cleft extending across both subdomains in which the trisaccharide molecule is oriented perpendicular to the UDP. Residues Glu(227), Asp(252), and Glu(281) dictate the binding orientation of the terminal Gal-2 moiety. Residue Glu(281) is in position to function as a catalytic base by deprotonating the incoming 3-hydroxyl group of the acceptor. The conserved DXD motif (Asp(194), Asp(195), Asp(196)) has direct interaction with the ribose of the UDP molecule as well as with the Mn(2+) ion. The key residues involved in substrate binding and catalysis are conserved in the glucuronyltransferase family as well as other glycosyltransferases.     

Distribution of mutations in human thymidylate synthase yielding resistance to 5-fluorodeoxyuridine

Thymidylate synthase (TS) catalyzes methylation of dUMP to dTMP and is the target of cancer chemotherapeutic agents (e.g. 5-fluorouracil). Here, we used error-prone PCR to mutagenize the full-length human TS cDNA and then selected mutants resistant to 5-fluorodeoxyuridine in a bacterial complementation system. We found that resistant mutants contained 1-5 amino acid substitutions and that these substitutions were located along the entire length of the polypeptide. Mutations were frequent near the active site Cys(195) and in the catalytically important Arg(50) loop; however, many mutations were also distributed throughout the remainder of the cDNA. Mutants containing a single amino acid replacement identified the following 14 residues as unreported sites of resistance: Glu(23), Thr(51), Thr(53), Val(84), Lys(93), Asp(110), Asp(116), Pro(194), Ser(206), Met(219), His(250), Asp(254), Tyr(258), and Lys(284). Many of these residues are distant from the active site and/or have no documented function in catalysis or resistance. We conclude that mutations distributed throughout the linear sequence and three-dimensional structure of human TS can confer resistance to 5-fluorodeoxyuridine. Our findings imply that long range interactions within proteins affect catalysis at the active site and that mutations at a distance can yield variant proteins with desired properties.     

Express analysis of protein amino acid sequences. Primary structure of Penicillium chrysogenum 152A guanyl-specific ribonuclease

The complete amino acid sequence of Penicillium chrysogenum 152A guanyl-specific RNase has been established using automated Edman degradation of two non-fractionated peptide mixtures produced by tryptic and staphylococcal protease digests of the protein. The RNase contains 102 amino acid residues: His2, Arg3, Asp7, Asn8, Thr5, Ser11, Glu4, Gln2, Pro4, Gly11, Ala13, Cys4, Val8, Ile3, Leu3, Tyr9, Phe5 (Mr 10 747).