Probing functional perfection in substructures of ribonuclease T1: double combinatorial random mutagenesis involving Asn43, Asn44, and Glu46 in the guanine binding loop

Combinatorial random mutageneses involving either Asn43 with Asn44 (set 1) or Glu46 with an adjacent insertion (set 2) were undertaken to explore the functional perfection of the guanine recognition loop of ribonuclease T(1) (RNase T(1)). Four hundred unique recombinants were screened in each set for their ability to enhance enzyme catalysis of RNA cleavage. After a thorough selection procedure, only six variants were found that were either as active or more active than wild type which included substitutions of Asn43 by Gly, His, Leu, or Thr, an unplanned Tyr45Ser substitution and Glu46Pro with an adjacent Glu47 insertion. Asn43His-RNase T(1) has the same loop sequence as that for RNases Pb(1) and Fl(2). None of the most active mutants were single substitutions at Asn44 or double substitutions at Asn43 and Asn44. A total of 13 variants were purified, and these were subjected to kinetic analysis using RNA, GpC, and ApC as substrates. Modestly enhanced activities with GpC and RNA involved both k(cat) and K(M) effects. Mutants having low activity with GpC had proportionately even lower relative activity with RNA. Asn43Gly-RNase T(1) and all five of the purified mutants in set 2 exhibited similar values of k(cat)/K(M) for ApC which were the highest observed and about 10-fold that for wild type. The specificity ratio [(k(cat)/K(M))(GpC)/(k(cat)/K(M))(ApC)] varied over 30 000-fold including a 10-fold increase [Asn43His variant; mainly due to a low (k(cat)/K(M))(ApC)] and a 3000-fold decrease (Glu46Ser/(insert)Gly47 variant; mainly due to a low (k(cat)/K(M))(GpC)) as compared with wild type. It is interesting that k(cat) (GpC) for the Tyr45Ser variant was almost 4-fold greater than for wild type and that Pro46/(insert)Glu47 RNase T(1) is 70-fold more active than the permuted variant (insert)Pro47-RNase T(1) which has a conserved Glu46. In any event, the observation that only 6 out of 800 variants surveyed had wild-type activity supports the view that functional perfection of the guanine recognition loop of RNase T(1) has been achieved.     

Deletion of the omega-loop in the active site of staphylococcal nuclease. 1. Effect on catalysis and stability

The high-resolution X-ray structure of wild-type staphylococcal nuclease (E43 SNase) suggests that Glu 43 acts a general basic catalyst to assist the attack of water on a phosphodiester substrate [Loll, P., & Lattman, E. E. (1989) Proteins: Struct., Funct., Genet. 5, 183]. Glu 43 is located at the base of the solvent-exposed and conformationally mobile omega-loop in the active site of E43 SNase having the sequence Glu43-Thr44-Lys45-His46-Pro47-Lys48- Lys49-Gly50-Val51-Glu52, where the gamma-carboxylate of Glu 52 is hydrogen bonded to the amide hydrogen of Glu 43. With a metabolic selection for SNase activity produced in an Escherichia coli host, we detected an unexpected deletion of residues 44-49 of the omega-loop of E43 SNase in cassette mutagenesis experiments designed to randomize codons 44 and 45 in the omega-loop and increase the activity of the previously described E43D mutation (D43 SNase). A high-resolution X-ray structure of D43 SNase has revealed that the E43D substitution significantly changes the structure of the omega-loop, reduces the interaction of the essential Ca2+ ion with its active-site ligands, and diminishes the network of hydrogen-bonded water molecules in the active site [Loll, P., & Lattman, E. E. (1990) Biochemistry 29, 6866]. This deletion of six amino acids from the omega-loop generates a protein (E43 delta SNase) having a partially solvent-exposed, surface beta-turn with the sequence Glu43-Gly50-Val51-Glu52; the structure of this beta-turn is addressed in the following article [Baldisseri et al. (1991) Biochemistry (following paper in this issue)].(ABSTRACT TRUNCATED AT 250 WORDS)     

His92Ala mutation in ribonuclease T1 induces segmental flexibility. An X-ray study.

In the genetically mutated ribonuclease T1 His92Ala (RNase T1 His92Ala), deletion of the active site His92 imidazole leads to an inactive enzyme. Attempts to crystallize RNase T1 His92Ala under conditions used for wild-type enzyme failed, and a modified protocol produced two crystal forms, one obtained with polyethylene glycol (PEG), and the other with phosphate as precipitants. Space groups are identical to wild-type RNase T1, P2(1)2(1)2(1), but unit cell dimensions differ significantly, associated with different molecular packings in the crystals; they are a = 31.04 A, b = 62.31 A, c = 43.70 A for PEG-derived crystals and a = 32.76 A, b = 55.13 A, c = 43.29 A for phosphate-derived crystals, compared to a = 48.73 A, b = 46.39 A, c = 41.10 A for uncomplexed wild-type RNase T1. The crystal structures were solved by molecular replacement and refined by stereochemically restrained least-squares methods based on Fo greater than or equal to sigma (Fo) of 3712 reflections in the resolution range 10 to 2.2 A (R = 15.8%) for the PEG-derived crystal and based on Fo greater than or equal to sigma (Fo) of 6258 reflections in the resolution range 10 to 1.8 A (R = 14.8%) for the phosphate-derived crystal. The His92Ala mutation deletes the hydrogen bond His92N epsilon H ... O Asn99 of wild-type RNase T1, thereby inducing structural flexibility and conformational changes in the loop 91 to 101 which is located at the periphery of the globular enzyme. This loop is stabilized in the wild-type protein by two beta-turns of which only one is retained in the crystals obtained with PEG. In the crystals grown with phosphate as precipitant, both beta-turns are deleted and the segment Gly94-Ala95-Ser96-Gly97 is so disordered that it is not seen at all. In addition, the geometry of the guanine binding site in both mutant studies is different from "empty" wild-type RNase T1 but similar to that found in complexes with guanosine derivatives: the Glu46 side-chain carboxylate hydrogen bonds to Tyr42 O eta; water molecules that are present in the guanine binding site of "empty" wild-type RNase T1 are displaced; the Asn43-Asn44 peptide is flipped such that phi/psi-angles of Asn44 are in alpha L-conformation (that is observed in wild-type enzyme when guanine is bound).(ABSTRACT TRUNCATED AT 400 WORDS)     

RNase T1 variant RV cleaves single-stranded RNA after purines due to specific recognition by the Asn46 side chain amide

Attempts to alter the guanine specificity of ribonuclease T1 (RNase T1) by rational or random mutagenesis have failed so far. The RNase T1 variant RV (Lys41Glu, Tyr42Phe, Asn43Arg, Tyr45Trp, and Glu46Asn) designed by combination of a random and a rational mutagenesis approach, however, exhibits a stronger preference toward adenosine residues than wild-type RNase T1. Steady state kinetics of the cleavage reaction of the two dinucleoside phosphate substrates adenylyl-3',5'-cytidine and guanylyl-3',5'-cytidine revealed that the ApC/GpC ratio of the specificity coefficient (k(cat)/K(m)) was increased approximately 7250-fold compared to that of the wild-type. The crystal structure of the nucleotide-free RV variant has been refined in space group P6(1) to a crystallographic R-factor of 19.9% at 1.7 A resolution. The primary recognition site of the RV variant adopts a similar conformation as already known from crystal structures of RNase T1 not complexed to any nucleotide. Noteworthy is a high flexibility of Trp45 and Asn46 within the three individual molecules in the asymmetric unit. In addition to the kinetic studies, these data indicate the participation of Asn46 in the specific recognition of the base and therefore a specific binding of adenosine.     

Cleavage of Rabbit Myelin Basic Protein by Plasmin: Isolation and Identification of the Major Products

Rabbit myelin basic protein (BP) was subjected to partial cleavage with plasmin, and 15 cleavage products were isolated by a combination of gel filtration and ion-exchange chromatography. Their identification was achieved by amino acid analysis and tryptic peptide mapping, supplemented in some instances by carboxy-terminal analyses with carboxypeptidases A, B, and Y and amino-terminal analyses with dipeptidyl aminopeptidase I. The results showed that major plasmic cleavage sites included the Lys89-Asn90, Lys133-Ser134, and Lys153-Leu154 bonds. Cleavages also occurred at the Arg31-His32, Lys53-Arg54, and Arg25-His26 bonds, but these appeared to be less extensive. A large number of additional peptides were produced in relatively low yield. The smaller of these were isolated from heterogeneous fractions by high-voltage electrophoresis-TLC. Amino acid analysis of these peptides showed that minor cleavage sites included the Arg9-His10, Lys13-Tyr14, Lys103-Gly104, Lys137-Gly138, Lys140-Gly141, and Arg160-Ser161 bonds. In spite of a lower selectivity toward peptide bonds in BP as compared with pepsin, cathepsin D, and thrombin, plasmin has the advantage over the former proteinases in that it does not cleave at or near the Phe44-Phe45 bond. Instead it cleaves at the Arg31-His32 and Lys53-Arg54 bonds, thus preserving the entire hydrophobic sequence Ile-Leu-Asp-Ser-Ile-Gly-Arg-Phe-Phe as well as short sequences to either side.     

Identifiying human MHC supertypes using bioinformatic methods

Classification of MHC molecules into supertypes in terms of peptide-binding specificities is an important issue, with direct implications for the development of epitope-based vaccines with wide population coverage. In view of extremely high MHC polymorphism (948 class I and 633 class II HLA alleles) the experimental solution of this task is presently impossible. In this study, we describe a bioinformatics strategy for classifying MHC molecules into supertypes using information drawn solely from three-dimensional protein structure. Two chemometric techniques-hierarchical clustering and principal component analysis-were used independently on a set of 783 HLA class I molecules to identify supertypes based on structural similarities and molecular interaction fields calculated for the peptide binding site. Eight supertypes were defined: A2, A3, A24, B7, B27, B44, C1, and C4. The two techniques gave 77% consensus, i.e., 605 HLA class I alleles were classified in the same supertype by both methods. The proposed strategy allowed "supertype fingerprints" to be identified. Thus, the A2 supertype fingerprint is Tyr(9)/Phe(9), Arg(97), and His(114) or Tyr(116); the A3-Tyr(9)/Phe(9)/Ser(9), Ile(97)/Met(97) and Glu(114) or Asp(116); the A24-Ser(9) and Met(97); the B7-Asn(63) and Leu(81); the B27-Glu(63) and Leu(81); for B44-Ala(81); the C1-Ser(77); and the C4-Asn(77).     

Quantitative analysis of the contribution of Glu46 and Asn98 to the guanosine specificity of ribonuclease T1

In the crystal structure of the ribonuclease T1 (RNase T1; EC 3.1.27.3)-2'-GMP complex the hydrogen-bonding potential of the guanine base is saturated [Arni, R., Heinemann, U., Tokuoka, R., & Saenger, W. (1988) J. Biol. Chem. 263, 15358-15368]. The oxygens of the Glu46 carboxylate and the Asn98 main-chain carbonyl act as hydrogen-bond acceptors for the N(1)H-C(2)-N(2)H2 part of the base. We measured the transesterification kinetics of wild-type and Glu46Ala RNase T1 using the GpU, IpU, and XpU series of analogous substrates. We found that the N(1)H---Glu46 O epsilon 1, the N(2)H---Glu46 O epsilon 2, and the N(2)H---Asn98 O hydrogen bonds have an apparent contribution of 2.7, 1.1, and 1.2 kcal/mol to the interaction energy of the enzyme and the transition state of the substrate. Wild-type RNase T1 discriminates guanine from nonionized xanthine (a guanine analogue in which the exocyclic amino group is replaced by an oxygen) by about 4.4 kcal/mol. Loss of the specific hydrogen bonds with the exocyclic amino group of the guanine base accounts for 2.4 kcal/mol of this discrimination energy; 2.0 kcal/mol is due to unfavorable non-H-bonded oxygen-oxygen contacts in the enzyme-xanthine complex. A pH dependence study shows that the deprotonated form of xanthine (i.e., the 6-keto-2-enolate anion; pKa = 5.4) is far less preferred, if not excluded, as substrate by wild-type RNase T1; this may be attributed to an electrostatic repulsion of the negatively charged xanthine by the Glu46 carboxylate group.     

Isolation and characterization of a proteinase from the sarcocarp of melon fruit.

A proteinase from the sarcocarp of melon (Cucumis Melo L. var. Prince) was purified by a three-step procedure involving batch-wise treatment with CM-cellulose fibers, column chromatography on CM-cellulose powder and gel filtration on Sephadex G-75. The final enzyme preparation was homogeneous on acrylamide gel electrophoresis. Its molecular weight was estimated by two different methods to be about 50,000. Anlayses indicated tha presence of 475 amino acid residues and at least 7 moles of hexose. The maximum activity was found in the alkaline pH region against casein as a substrate. The optimum temperature against casein was 70 degrees at pH 7.1. The enzyme was strongly inhibited by diisopropyl fluorophosphate, partly inhibited by HgCl2 and not inhibited by EDTA, p-chloromercuribenzoic acid, N-tosyl-L-lysine chloromethyl ketone, N-tosyl-L-phenylalanine chloromethyl ketone, and soybean trypsin inhibitor. The reduced and carboxymethylated insulin B-chain was cleaved at the peptide bonds of Asn3-Gln4, Cm-Cys7-Gly8, Glu13-Ala14, Leu15-Tyr16, Cm-Cys19-Gly20, Phe25-Tyr26, Pro28-Lys29, and Lys29-Ala30 by the enzyme.     

Purification of a new extracellular 90-kDa serine proteinase with isoelectric point of 3.9 from Bacillus subtilis (natto) and elucidation of its distinct mode of action.

A new extracellular 90-kDa serine proteinase with an isoelectric point (pI) of 3.9 was purified from Bicillus subtilis (natto). Microheterogeneity was detected in the 50-kDa protease of bacillopeptidase F with pI 4.4 reported previously by Wu et al. and the sequence for the first 25 amino acids in the internal region of the enzyme was analyzed: ATDGVEWNVDQIDAPKAWALGYDGA. The cleavage sites in the oxidized B-chain of insulin by the proteinase were CySO3H7-Gly8, Val12-Glu13, Try16-Leu17, and Phe25-Tyr26. The activity was inhibited by phenylmethylsulfonyl fluoride (PMSF) and chymostatin, while the activity was not inhibited by proteinaceous Streptomyces subtilisin inhibitor (SSI) or alpha 2-macroglubulin.     

The influence of ATP on the binding of aromatic amino acids to the ligand response domain of the tyrosine repressor of Haemophilus influenzae

Mitogillin and related fungal ribotoxins are small basic ribonucleolytic proteins that inhibit protein synthesis by specifically hydrolyzing a single phosphodiester bond in the universally conserved alpha-sarcin/ricin loop (SRL) of large subunit ribosomal RNAs. It was previously shown that mitogillin is a natural derivative of a T1/U2-like ribonuclease with inserted domains that are involved in target selection and specificity. Site-directed mutagenesis was used to substitute single amino acids in the previously identified functional domains Ala1-Tyr24 (B1-L1-B2 domain) and Lys106-Lys113 (L4 region). Examination of the activities of the mutants in the digestion of polyinosinic acid (a ribonuclease substrate) and specific cleavage of the SRL shows that Asn7Ala and Lys111Gln substitutions lead to altered ribonuclease activity and diminished substrate specificity consistent with the proposed functions of these domains.     

Photoactivation perturbs the membrane-embedded contacts between sensory rhodopsin II and its transducer

The photoactivation mechanism of the sensory rho-dopsin II (SRII)-HtrII receptor-transducer complex of Natronomonas pharaonis was investigated by time-resolved Fourier transform infrared difference spectroscopy to identify structural changes associated with early events in the signal relay mechanism from the receptor to the transducer. Several prominent bands in the wild-type SRII-HtrII spectra are affected by amino acid substitutions at the receptor Tyr(199) and transducer Asn(74) residues, which form a hydrogen bond between the two proteins near the middle of the bilayer. Our results indicate disappearance of this hydrogen bond in the M and O photointermediates, the likely signaling states of the complex. This event represents one of the largest light-induced alterations in the binding contacts between the receptor and transducer. The vibrational frequency changes suggest that Asn(74) and Tyr(199) form other stronger hydrogen bonds in the M state. The light-induced disruption of the Tyr(199)-Asn(74) bond also occurs when the Schiff base counterion Asp(75) is replaced with a neutral asparagine. We compared the decrease in intensity of difference bands assigned to the Tyr(199)-Asn(74) pair and to chromophore and protein groups of the receptor at various time points during the recovery of the initial state. All difference bands exhibit similar decay kinetics indicating that reformation of the Tyr(199)-Asn(74) hydrogen bond occurs concomitantly with the decay of the M and O photointermediates. This work demonstrates that the signal relay from SRII to HtrII involves early structural alterations in the deeply membrane-embedded domain of the complex and provides a spectroscopic signal useful for correlation with the downstream events in signal transduction.     

RNase T1 mutant Glu46Gln binds the inhibitors 2'GMP and 2'AMP at the 3' subsite.

On the basis of molecular dynamics and free-energy perturbation approaches, the Glu46Gln (E46Q) mutation in the guanine-specific ribonuclease T1 (RNase T1) was predicted to render the enzyme specific for adenine. The E46Q mutant was genetically engineered and characterized biochemically and crystallographically by investigating the structures of its two complexes with 2'AMP and 2'GMP. The ribonuclease E46Q mutant is nearly inactive towards dinucleoside phosphate substrates but shows 17% residual activity towards RNA. It binds 2'AMP and 2'GMP equally well with dissociation constants of 49 microM and 37 microM, in contrast to the wild-type enzyme, which strongly discriminates between these two nucleotides, yielding dissociation constants of 36 microM and 0.6 microM. These data suggest that the E46Q mutant binds the nucleotides not to the specific recognition site but to the subsite at His92. This was confirmed by the crystal structures, which also showed that the Gln46 amide is hydrogen bonded to the Phe100 N and O atoms, and tightly anchored in this position. This interaction may either have locked the guanine recognition site so that 2'AMP and 2'GMP are unable to insert, or the contribution to guanine recognition of Glu46 is so important that the E46Q mutant is unable to function in recognition of either guanine and adenine.     

Characterization of the urinary metabolites of dipetarudin

Dipetarudin is a hybrid thrombin inhibitor composed of the N-terminal structure of dipetalogastin II and the exosite 1 blocking segment of hirudin. Pharmacokinetic studies demonstrated that it distributes in extravascular and intravascular spaces and is exclusively eliminated by the kidneys. Two active metabolites of dipetarudin with molecular masses of 6142 and 5395 Da, respectively, were isolated from rat urine. Analysis of their N-terminal sequences and molecular masses demonstrated that dipetarudin is cleaved in a first step at the peptide bond Phe55-Glu56 and then, at Gly3-Asn4. Nonmetabolized dipetarudin was not found in rat urine. Proteases localized in the proximal tubulus cells of kidneys might be responsible for its degradation.     

Crystallographic characterization of wild-type and mutant ribonuclease T1 complexes with several ribonucleotides

Ribonuclease T1 and the mutant enzymes were cocrystallized with several ribonucleotides, including non-hydrolyzable substrate analogs of di- and triribonucleotides, which have a novel guanylate in which the 2'-hydroxyl group of the ribose is replaced by a fluorine atom. One of the mutant enzymes has a tryptophan residue, instead of Tyr45 of the wild-type enzyme, to enhance the binding of ribonucleotides to the enzyme and the other mutant enzyme has histidine and aspartate residues, instead of Asn43 and Asn44, respectively, to reproduce the natural substitutions found in ribonuclease Ms. Polymorphism of the crystals was observed for wild-type and mutant enzymes. However, orthorhombic crystals, which are virtually all isomorphous to each other, were successfully obtained from wild-type and mutant (Y45W) enzymes by the macroscopic seeding technique using mother crystals of the wild-type ribonuclease T1 complexed with 2'GMP or 3'GMP. The diffraction patterns of these crystals extend beyond 2.5 A resolution and the diffraction data were collected from some of the crystals on a diffractometer up to a range of 2.5 to 1.8 A resolution.     

From structure to function: insights into the catalytic substrate specificity and thermostability displayed by Bacillus subtilis mannanase BCman

BCman, a β-mannanase from the plant root beneficial bacterium Bacillus subtilis Z-2, has a potential to be used in the production of mannooligosaccharide, which shows defense induction activity on both melon and tobacco, and plays an important role in the biological control of plant disease. Here we report the biochemical properties and crystal structure of BCman-GH26 enzyme. Kinetic analysis reveals that BCman is an endo-β-mannanase, specific for mannan, and has no activity on mannooligosaccharides. The catalytic acid/base Glu167 and nucleophile Glu266 are positioned on the β4 and β7 strands, respectively. The 1.45-Å crystal structure reveals that BCman is a typical (β/α)₈ folding type. One large difference from the saddle-shaped active center of other endo-β-mannanases is the presence of a shallow-dish-shaped active center and substrate-binding site that are both unique to BCman. These differences are mainly due to important changes in the length and position of loop 1 (Phe37-Met47), loop 2 (Ser103-Ala134), loop3 (Phe162-Asn185), loop 4 (Tyr215-Ile236), loop 5 (Pro269-Tyr278), and loop 6 (Trp298-Gly309), all of which surround the active site. Data from isothermal titration calorimetry and crystallography indicated only two substrate-binding subsites (+ 1 and − 1) within the active site of BCman. These two sites are involved in the enzyme's mannan degradation activity and in restricting the binding capacity for mannooligosaccharides. Binding and catalysis of BCman to mannan is mediated mainly by a surface containing a strip of solvent-exposed aromatic rings of Trp302, Trp298, Trp172, and Trp72. Additionally, BCman contains a disulfide bond (Cys66Cys86) and a special His1-His23-Glu336 metal-binding site. This secondary structure is a key factor in the enzyme's stability.     

Construction of chimeric glucansucrases for analyzing substrate-binding regions that affect the structure of glucan products

A gene that encodes dextransucrase S (dsrS) from Leuconostoc mesenteroides NRRL B-512F encodes a glucansucrase dextransucrase S (DSRS) which mainly produces water-soluble glucan (dextran), while the dsrT5 gene derived from dsrT of the B-512F strain encodes an enzyme dextransucrase T5 (DSRT5), which mainly produces water-insoluble glucan. Tyr340-Asn510 of DSRS and Tyr307-Asn477 of DSRT5 (Site 1), Lys696-Gly768 of DSRS and Lys668-Gly740 of DSRT5 (Site 2), and Asn917-Lys1131 of DSRS and Asn904-Lys1118 of DSRT5 (Site 3) were exchanged and six different chimeric enzymes were constructed. Water-soluble glucan produced by recombinant DSRS was composed of 64% 6-linked glucopyranoside (Glcp), 9% 3,6-linked Glcp, and 13% 4-linked Glcp. Water-insoluble glucan produced by recombinant DSRT5 was composed of 47% 6-linked Glcp and 43% 3-linked Glcp. All of the chimeric enzymes produced glucans different from the ones produced by their parental enzymes. Some of the glucans produced by chimeric enzymes were extremely changed. The Site 1 chimeric enzyme of DSRS (STS1) produced water-soluble glucan composed mostly of 6-linked Glcp. That of DSRT5 (TST1) produced water-insoluble glucan composed mostly of 4-linked Glcp. The Site 3 chimeric enzyme of DSRS (STS3) produced mainly water-insoluble glucan, DSRT5 (TST3) produced mainly water-soluble glucans, and all of the glucan fractions consisted of 3-Glcp, 4-Glcp, and 6-Glcp. The amounts of the three linkages in the water-soluble glucan produced by TST3 were about 1:1:1. Site 1 was assumed to be important for making or avoiding making alpha-1,4 linkages, while Site 3 was assumed to be important for determining the kinds of glucosyl linkages made.     

Assembly and ligand binding properties of the water-soluble extracellular domains of the glutamate receptor 1 subunit

High resolution structural studies of models of glutamate receptors (GluRs) have been limited to monomeric models of the ligand-binding site. To obtain oligomeric models of glutamate receptors that can reveal more complete structural information, we examined the assembly and ligand binding properties of two truncated versions of the GluR1 subunit. The first version, GluR1-WS, consisted of only the N-terminal extracellular segment (Ala(1)-Glu(520)) bridged by a synthetic linker to the second extracellular domain (Asn(615)-Gly(790)). The second version, GluR1-M1, consisted of the first N-terminal extracellular domain (Ala(1)-Glu(520)) bridged by a synthetic linker to a second segment containing the second extracellular domain, the third transmembrane domain, and the intracellular C-terminal domain (Asn(615)-Leu(889)). When expressed in Xenopus oocytes, GluR-WS was secreted and water-soluble; GluR1-M1 was displayed on the surface of oocytes. GluR1-WS exhibited a velocity sedimentation profile that was consistent with assembly of homooligomers and bound the glutamate receptor agonist alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid with high affinity. These findings show that the extracellular domains of GluR1 that are sufficient for ligand binding apparently are sufficient for subunit assembly and might be a suitable target for structural studies of a water-soluble GluR1 oligomer.     

Role of interactions involving C-terminal nonpolar residues of hirudin in the formation of the thrombin-hirudin complex

The role of interactions involving C-terminal nonpolar residues of hirudin in the formation of the thrombin-hirudin complex has been investigated by site-directed mutagenesis. The residues Phe56, Pro60, and Tyr63 of hirudin were replaced by a number of different amino acids, and the kinetics of the inhibition of thrombin by the mutant proteins were determined. Phe56 could be replaced by aromatic amino acids without significant loss in binding energy. While substitution of Phe56 by alanine decreased the binding energy (delta G degrees b by only 1.9 kJ mol-1, replacement of this residue by amino acids with branched side chains caused larger decreases in delta G degrees b. For example, the mutant Phe56----Val displayed a decrease in delta G degrees b of 10.5 kJ mol-1. Substitution of Pro60 by alanine or glycine resulted in a decrease in delta G degrees b of about 6 kJ mol-1. Tyr63 could be replaced by phenylalanine without any loss in binding energy, and replacement of this residue by alanine caused a decrease of 2.2 kJ mol-1 in delta G degrees b. Substitution of Tyr63 by residues with branched side chains resulted in smaller decreases in delta G degrees b than those seen with the corresponding substitutions of Phe56; for example, the mutant Tyr63----Val showed a decrease in binding energy of 5.1 kJ mol-1. The effects of the mutations are discussed in terms of the crystal structure of the thrombin-hirudin complex.     

Effects of site-directed mutagenesis of the loop residue of the N-terminal domain Gly117 of thermolysin on its catalytic activity

In the N-terminal domain of thermolysin, two polypeptide strands, Asn112-Ala113-Phe114-Trp115 and Ser118-Gln119-Met120-Val121-Tyr122, are connected by a short loop, Asn116-Gly117, to form an anti-parallel β-sheet. The Asn112-Trp115 strand is located in the active site, while the Ser118-Tyr122 strand and the Asn116-Gly117 loop are located outside the active site. In this study, we explored the catalytic role of Gly117 by site-directed mutagenesis. Five variants, G117A (Gly117 is replaced by Ala), G117D, G117E, G117K, and G117R, were produced by co-expressing in Escherichia coli the mature and pro domains as independent polypeptides. The production levels were in the order G117E > wild type > G117K, G117R > G117D. G117A was hardly produced. This result is in contrast to our previous one that all 72 active-site thermolysin variants were produced at the similar levels whether they retained activity or not (M. Kusano et al. J. Biochem., 145, 103-113 (2009)). G117E exhibited lower activity in the hydrolysis of N-[3-(2-furyl)acryloyl]-glycyl-L-leucine amide and higher activity in the hydrolysis of N-carbobenzoxy-L-aspartyl-L-phenylalanine methyl ester than the wild-type thermolysin. G117K and G117R exhibited considerably reduced activities. This suggests that Gly117 plays an important role in the activity and stability of thermolysin, presumably by affecting the geometries of the Asn112-Trp115 and Ser118-Tyr122 strands.     

Crystal structure of guanosine-free ribonuclease T1, complexed with vanadate (V), suggests conformational change upon substrate binding

Ribonuclease T1 was crystallized in the presence of vanadate(V). The crystal structure was solved by molecular replacement and refined by least-squares methods using stereochemical restraints. The refinement was based on data between 10 and 1.8 A and converged at a crystallographic R factor of 0.137. Except for the substrate-recognition site the three-dimensional structure of ribonuclease T1 closely resembles the structure of the enzyme complexed with guanosine 2'-phosphate and its derivatives. A tetrahedral anion was found at the catalytic site and identified as H2VO4-. This is the first crystal structure of ribonuclease T1 determined in the absence of bound substrate analogue. Distinct structural differences between guanosine-free and complexed ribonuclease T1 are observed at the base-recognition site: The side chains of Tyr45 and Glu46 and the region around Asn98 changed their conformations, and the peptide bond between Asn43 and Asn44 has turned around by 140 degrees. We suggest that the structural differences seen in the crystal structures of free and complexed ribonuclease T1 are related to conformational adjustments associated with the substrate binding process.