Crystal structures of HIV protease V82A and L90M mutants reveal changes in the indinavir-binding site.

The crystal structures of the wild-type HIV-1 protease (PR) and the two resistant variants, PR(V82A) and PR(L90M), have been determined in complex with the antiviral drug, indinavir, to gain insight into the molecular basis of drug resistance. V82A and L90M correspond to an active site mutation and nonactive site mutation, respectively. The inhibition (K(i)) of PR(V82A) and PR(L90M) was 3.3- and 0.16-fold, respectively, relative to the value for PR. They showed only a modest decrease, of 10-15%, in their k(cat)/K(m) values relative to PR. The crystal structures were refined to resolutions of 1.25-1.4 A to reveal critical features associated with inhibitor resistance. PR(V82A) showed local changes in residues 81-82 at the site of the mutation, while PR(L90M) showed local changes near Met90 and an additional interaction with indinavir. These structural differences concur with the kinetic data.     

The L76V drug resistance mutation decreases the dimer stability and rate of autoprocessing of HIV-1 protease by reducing internal hydrophobic contacts

The mature HIV-1 protease (PR) bearing the L76V drug resistance mutation (PR(L76V)) is significantly less stable, with a >7-fold higher dimer dissociation constant (K(d)) of 71 ± 24 nM and twice the sensitivity to urea denaturation (UC(50) = 0.85 M) relative to those of PR. Differential scanning calorimetry showed decreases in T(m) of 12 °C for PR(L76V) in the absence of inhibitors and 5-7 °C in the presence of inhibitors darunavir (DRV), saquinavir (SQV), and lopinavir (LPV), relative to that of PR. Isothermal titration calorimetry gave a ligand dissociation constant of 0.8 nM for DRV, ～160-fold higher than that of PR, consistent with DRV resistance. Crystal structures of PR(L76V) in complexes with DRV and SQV were determined at resolutions of 1.45-1.46 ?. Compared to the corresponding PR complexes, the mutated Val76 lacks hydrophobic interactions with Asp30, Lys45, Ile47, and Thr74 and exhibits closer interactions with Val32 and Val56. The bound DRV lacks one hydrogen bond with the main chain of Asp30 in PR(L76V) relative to PR, possibly accounting for the resistance to DRV. SQV shows slightly improved polar interactions with PR(L76V) compared to those with PR. Although the L76V mutation significantly slows the N-terminal autoprocessing of the precursor TFR-PR(L76V) to give rise to the mature PR(L76V), the coselected M46I mutation counteracts the effect by enhancing this rate but renders the TFR-PR(M46I/L76V) precursor less responsive to inhibition by 6 μM LPV while preserving inhibition by SQV and DRV. The correlation of lowered stability, higher K(d), and impaired autoprocessing with reduced internal hydrophobic contacts suggests a novel molecular mechanism for drug resistance.     

Selection of Drug-Resistant Feline Immunodeficiency Virus (FIV) Encoding FIV/HIV Chimeric Protease in the Presence of HIV-Specific Protease Inhibitors

An infectious chimeric feline immunodeficiency virus (FIV)/HIV strain carrying six HIV-like protease (PR) mutations (I37V/N55M/V59I/I98S/Q99V/P100N) was subjected to selection in culture against the PR inhibitor lopinavir (LPV), darunavir (DRV), or TL-3. LPV selection resulted in the sequential emergence of V99A (strain S-1X), I59V (strain S-2X), and I108V (strain S-3X) mutations, followed by V37I (strain S-4X). Mutant PRs were analyzed in vitro, and an isogenic virus producing each mutant PR was analyzed in culture for LPV sensitivity, yielding results consistent with the original selection. The 50% inhibitory concentrations (IC₅₀s) for S-1X, S-2X, S-3X, and S-4X were 95, 643, 627, and 1,543 nM, respectively. The primary resistance mutations, V99⁸²A, I59⁵⁰V, and V37³²I, are consistent with the resistance pattern developed by HIV-1 under similar selection conditions. While resistance to LPV emerged readily, similar PR mutations causing resistance to either DRV or TL-3 failed to emerge after passage for more than a year. However, a G37D mutation in the nucleocapsid (NC) was observed in both selections and an isogenic G37D mutant replicated in the presence of 100 nM DRV or TL-3, whereas parental chimeric FIV could not. An additional mutation, L92V, near the PR active site in the folded structure recently emerged during TL-3 selection. The L92V mutant PR exhibited an IC₅₀ of 50 nM, compared to 35 nM for 6s-98S PR, and processed the NC-p2 junction more efficiently, consistent with increased viral fitness. These findings emphasize the role of mutations outside the active site of PR in increasing viral resistance to active-site inhibitors and suggest additional targets for inhibitor development.     

Quantitative chimeric analysis of six specificity determinants that differentiate Escherichia coli aspartate from tyrosine aminotransferase

The six mutations, referred to as the Hex mutations, that together have been shown to convert Escherichia coli aspartate aminotransferase (AATase) specificity to be substantially like that of E. coli tyrosine aminotransferase (TATase) are dissected into two groups, (T109S/N297S) and (V39L/K41Y/T47I/N69L). The letters on the left and right of the numbers designate AATase and TATase residues, respectively. The T109S/N297S pair has been investigated previously. The latter group, the "Grease" set, is now placed in the AATase framework, and the retroGrease set (L39V/Y41K/I47T/L69N) is substituted into TATase. The Grease mutations in the AATase framework were found primarily to lower K(M)s for both aromatic and dicarboxylic substrates. In contrast, retroGrease TATase exhibits lowered k(cat)s for both substrates. The six retroHex mutations, combining retroGrease and S109T/S297N, were found to invert the substrate specificity of TATase, creating an enzyme with a nearly ninefold preference (k(cat)/K(M)) for aspartate over phenylalanine. The retroHex mutations perturb the electrostatic environment of the pyridoxal phosphate cofactor, as evidenced by a spectrophotometric titration of the internal aldimine, which uniquely shows two pK(a)s, 6.1 and 9.1. RetroHex was also found to have impaired dimer stability, with a K(D) for dimer dissociation of 350 nM compared with the wild type K(D) of 4 nM. Context dependence and additivity analyses demonstrate the importance of interactions of the Grease residues with the surrounding protein framework in both the AATase and TATase contexts, and with residues 109 and 297 in particular. Context dependence and cooperativity are particularly evident in the effects of mutations on k(cat)/K(M)(Asp). Effects on k(cat)/K(M)(Phe) are more nearly additive and context independent.     

Enzymatic and structural analysis of the I47A mutation contributing to the reduced susceptibility to HIV protease inhibitor lopinavir

Lopinavir (LPV) is a second-generation HIV protease inhibitor (PI) designed to overcome resistance development in patients undergoing long-term antiviral therapy. The mutation of isoleucine at position 47 of the HIV protease (PR) to alanine is associated with a high level of resistance to LPV. In this study, we show that recombinant PR containing a single I47A substitution has the inhibition constant (K(i) ) value for lopinavir by two orders of magnitude higher than for the wild-type PR. The addition of the I47A substitution to the background of a multiply mutated PR species from an AIDS patient showed a three-order-of-magnitude increase in K(i) in vitro relative to the patient PR without the I47A mutation. The crystal structure of I47A PR in complex with LPV showed the loss of van der Waals interactions in the S2/S2' subsites. This is caused by the loss of three side-chain methyl groups due to the I47A substitution and by structural changes in the A47 main chain that lead to structural changes in the flap antiparallel beta-strand. Furthermore, we analyzed possible interaction of the I47A mutation with secondary mutations V32I and I54V. We show that both mutations in combination with I47A synergistically increase the relative resistance to LPV in vitro. The crystal structure of the I47A/I54V PR double mutant in complex with LPV shows that the I54V mutation leads to a compaction of the flap, and molecular modeling suggests that the introduction of the I54V mutation indirectly affects the strain of the bound inhibitor in the PR binding cleft.     

Structure and inhibition of a quorum sensing target from Streptococcus pneumoniae

Streptococcus pneumoniae 5'-methylthioadenosine/S-adenosylhomocysteine hydrolase (MTAN) catalyzes the hydrolytic deadenylation of its substrates to form adenine and 5-methylthioribose or S-ribosylhomocysteine (SRH). MTAN is not found in mammals but is involved in bacterial quorum sensing. MTAN gene disruption affects the growth and pathogenicity of bacteria, making it a target for antibiotic design. Kinetic isotope effects and computational studies have established a dissociative S(N)1 transition state for Escherichia coli MTAN, and transition state analogues resembling the transition state are powerful inhibitors of the enzyme [Singh, V., Lee, J. L., Nú?ez, S., Howell, P. L., and Schramm, V. L. (2005) Biochemistry 44, 11647-11659]. The sequence of MTAN from S. pneumoniae is 40% identical to that of E. coli MTAN, but S. pneumoniae MTAN exhibits remarkably distinct kinetic and inhibitory properties. 5'-Methylthio-Immucillin-A (MT-ImmA) is a transition state analogue resembling an early S(N)1 transition state. It is a weak inhibitor of S. pneumoniae MTAN with a K(i) of 1.0 microM. The X-ray structure of S. pneumoniae MTAN with MT-ImmA indicates a dimer with the methylthio group in a flexible hydrophobic pocket. Replacing the methyl group with phenyl (PhT-ImmA), tolyl (p-TolT-ImmA), or ethyl (EtT-ImmA) groups increases the affinity to give K(i) values of 335, 60, and 40 nM, respectively. DADMe-Immucillins are geometric and electrostatic mimics of a fully dissociated transition state and bind more tightly than Immucillins. MT-DADMe-Immucillin-A inhibits with a K(i) value of 24 nM, and replacing the 5'-methyl group with p-Cl-phenyl (p-Cl-PhT-DADMe-ImmA) gave a K(i) value of 0.36 nM. The inhibitory potential of DADMe-Immucillins relative to the Immucillins supports a fully dissociated transition state structure for S. pneumoniae MTAN. Comparison of active site contacts in the X-ray crystal structures of E. coli and S. pneumoniae MTAN with MT-ImmA would predict equal binding, yet most analogues bind 10(3)-10(4)-fold more tightly to the E. coli enzyme. Catalytic site efficiency is primarily responsible for this difference since k(cat)/K(m) for S. pneumoniae MTAN is decreased 845-fold relative to that of E. coli MTAN.     

Structural and kinetic analysis of drug resistant mutants of HIV-1 protease.

Mutants of HIV-1 protease that are commonly selected on exposure to different drugs, V82S, G48V, N88D and L90M, showed reduced catalytic activity compared to the wild-type protease on cleavage site peptides, CA-p2, p6pol-PR and PR-RT, critical for viral maturation. Mutant V82S is the least active (2-20% of wild-type protease), mutants N88D, R8Q, and L90M exhibit activities ranging from 20 to 40% and G48V from 50 to 80% of the wild-type activity. In contrast, D30N is variable in its activity on different substrates (10-110% of wild-type), with the PR-RT site being the most affected. Mutants K45I and M46L, usually selected in combination with other mutations, showed activities that are similar to (60-110%) or greater than (110-530%) wild-type, respectively. No direct relationship was observed between catalytic activity, inhibition, and structural stability. The mutants D30N and V82S were similar to wild-type protease in their stability toward urea denaturation, while R8Q, G48V, and L90M showed 1.5 to 2.7-fold decreased stability, and N88D and K45I showed 1.6 to 1.7-fold increased stability. The crystal structures of R8Q, K45I and L90M mutants complexed with a CA-p2 analog inhibitor were determined at 2.0, 1.55 and 1.88 A resolution, respectively, and compared to the wild-type structure. The intersubunit hydrophobic contacts observed in the crystal structures are in good agreement with the relative structural stability of the mutant proteases. All these results suggest that viral resistance does not arise by a single mechanism.     

Engineering Streptomyces clavuligerus deacetoxycephalosporin C synthase for optimal ring expansion activity toward penicillin G

The deacetoxycephalosporin C synthase (DAOCS) from Streptomyces clavuligerus was engineered with the aim of enhancing the conversion of penicillin G into phenylacetyl-7-aminodeacetoxycephalosporanic acid, a precursor of 7-aminodeacetoxycephalosporanic acid, for industrial application. A single round of random mutagenesis followed by the screening of 5,500 clones identified three mutants, G79E, V275I, and C281Y, that showed a two- to sixfold increase in the k(cat)/K(m) ratio compared to the wild-type enzyme. Site-directed mutagenesis to modify residues surrounding the substrate resulted in three mutants, N304K, I305L, and I305M, with 6- to 14-fold-increased k(cat)/K(m) values. When mutants containing all possible combinations of these six sites were generated to optimize the ring expansion activity for penicillin G, the double mutant, YS67 (V275I, I305M), showed a significant 32-fold increase in the k(cat)/K(m) ratio and a 5-fold increase in relative activity for penicillin G, while the triple mutant, YS81 (V275I, C281Y, I305M), showed an even greater 13-fold increase in relative activity toward penicillin G. Our results demonstrate that this is a robust approach to the modification of DAOCS for an optimized DAOCS-penicillin G reaction.     

Effect of flap mutations on structure of HIV-1 protease and inhibition by saquinavir and darunavir

HIV-1 (human immunodeficiency virus type 1) protease (PR) and its mutants are important antiviral drug targets. The PR flap region is critical for binding substrates or inhibitors and catalytic activity. Hence, mutations of flap residues frequently contribute to reduced susceptibility to PR inhibitors in drug-resistant HIV. Structural and kinetic analyses were used to investigate the role of flap residues Gly48, Ile50, and Ile54 in the development of drug resistance. The crystal structures of flap mutants PR_I50V (PR with I50V mutation), PR_I54V (PR with I54V mutation), and PR_I54M (PR with I54M mutation) complexed with saquinavir (SQV) as well as PR_G48V (PR with G48V mutation), PR_I54V, and PR_I54M complexed with darunavir (DRV) were determined at resolutions of 1.05-1.40 Å. The PR mutants showed changes in flap conformation, interactions with adjacent residues, inhibitor binding, and the conformation of the 80s loop relative to the wild-type PR. The PR contacts with DRV were closer in PR_G48V-DRV than in the wild-type PR-DRV, whereas they were longer in PR_I54M-DRV. The relative inhibition of PR_I54V and that of PR_I54M were similar for SQV and DRV. PR_G48V was about twofold less susceptible to SQV than to DRV, whereas the opposite was observed for PR_I50V. The observed inhibition was in agreement with the association of G48V and I50V with clinical resistance to SQV and DRV, respectively. This analysis of structural and kinetic effects of the mutants will assist in the development of more effective inhibitors for drug-resistant HIV.     

Lack of synergy for inhibitors targeting a multi-drug-resistant HIV-1 protease

The three-dimensional structures of indinavir and three newly synthesized indinavir analogs in complex with a multi-drug-resistant variant (L63P, V82T, I84V) of HIV-1 protease were determined to approximately 2.2 A resolution. Two of the three analogs have only a single modification of indinavir, and their binding affinities to the variant HIV-1 protease are enhanced over that of indinavir. However, when both modifications were combined into a single compound, the binding affinity to the protease variant was reduced. On close examination, the structural rearrangements in the protease that occur in the tightest binding inhibitor complex are mutually exclusive with the structural rearrangements seen in the second tightest inhibitor complex. This occurs as adaptations in the S1 pocket of one monomer propagate through the dimer and affect the conformation of the S1 loop near P81 of the other monomer. Therefore, structural rearrangements that occur within the protease when it binds to an inhibitor with a single modification must be accounted for in the design of inhibitors with multiple modifications. This consideration is necessary to develop inhibitors that bind sufficiently tightly to drug-resistant variants of HIV-1 protease to potentially become the next generation of therapeutic agents.     

Improvement of oxidative and thermostability of N-carbamyl-d-amino Acid amidohydrolase by directed evolution

N-Carbamyl-D-amino acid amidohydrolase (N-carbamoylase), which is currently employed in the industrial production of unnatural D-amino acid in conjunction with D-hydantoinase, has low oxidative and thermostability. We attempted the simultaneous improvement of the oxidative and thermostability of N-carbamoylase from Agrobacterium tumefaciens NRRL B11291 by directed evolution using DNA shuffling. In a second generation of evolution, the best mutant 2S3 with improved oxidative and thermostability was selected, purified and characterized. The temperature at which 50% of the initial activity remains after incubation for 30 min was 73 degrees C for 2S3, whereas it was 61 degrees C for wild-type enzyme. Treatment of wild-type enzyme with 0.2 mM hydrogen peroxide for 30 min at 25 degrees C resulted in a complete loss of activity, but 2S3 retained about 79% of the initial activity under the same conditions. The K(m) value of 2S3 was estimated to be similar to that of wild-type enzyme; however k(cat) was decreased, leading to a slightly reduced value of k(cat)/K(m), compared with wild-type enzyme. DNA sequence analysis revealed that six amino acid residues were changed in 2S3 and substitutions included Q23L, V40A, H58Y, G75S, M184L and T262A. The stabilizing effects of each amino acid residue were investigated by incorporating mutations individually into wild-type enzyme. Q23L, H58Y, M184L and T262A were found to enhance both oxidative and thermostability of the enzyme and of them, T262A showed the most significant effect. V40A and G75S gave rise to an increase only in oxidative stability. The positions of the mutated amino acid residues were identified in the structure of N-carbamoylase from Agrobacterium sp. KNK 712 and structural analysis of the stabilizing effects of each amino acid substitution was also carried out.     

A synergy of activity,stability, and inhibitor‐interaction of HIV‐1 protease mutants evolved under drug‐pressure

A clinically‐relevant, drug‐resistant mutant of HIV‐1 protease (PR), termed Flap+_(I54V) and containing L10I, G48V, I54V and V82A mutations, is known to produce significant changes in the entropy and enthalpy balance of drug‐PR interactions, compared to wild‐type PR. A similar mutant, Flap+_(I54A), which evolves from Flap+_(I54V) and contains the single change at residue 54 relative to Flap+_(I54V), does not. Yet, how Flap+_(I54A) behaves in solution is not known. To understand the molecular basis of V54A evolution, we compared nuclear magnetic resonance (NMR) spectroscopy, fluorescence spectroscopy, isothermal titration calorimetry, and enzymatic assay data from four PR proteins: PR (pWT), Flap+_(I54V), Flap+_(I54A), and Flap+_(I54), a control mutant that contains only L10I, G48V and V82A mutations. Our data consistently show that selection to the smaller side chain at residue 54, not only decreases inhibitor affinity, but also restores the catalytic activity.     

Gene cloning and molecular characterization of lysine decarboxylase from Selenomonas ruminantium delineate its evolutionary relationship to ornithine decarboxylases from eukaryotes

Lysine decarboxylase (LDC; EC 4.1.1.18) from Selenomonas ruminantium comprises two identical monomeric subunits of 43 kDa and has decarboxylating activities toward both L-lysine and L-ornithine with similar K(m) and V(max) values (Y. Takatsuka, M. Onoda, T. Sugiyama, K. Muramoto, T. Tomita, and Y. Kamio, Biosci. Biotechnol. Biochem. 62:1063-1069, 1999). Here, the LDC-encoding gene (ldc) of this bacterium was cloned and characterized. DNA sequencing analysis revealed that the amino acid sequence of S. ruminantium LDC is 35% identical to those of eukaryotic ornithine decarboxylases (ODCs; EC 4.1.1.17), including the mouse, Saccharomyces cerevisiae, Neurospora crassa, Trypanosoma brucei, and Caenorhabditis elegans enzymes. In addition, 26 amino acid residues, K69, D88, E94, D134, R154, K169, H197, D233, G235, G236, G237, F238, E274, G276, R277, Y278, K294, Y323, Y331, D332, C360, D361, D364, G387, Y389, and F397 (mouse ODC numbering), all of which are implicated in the formation of the pyridoxal phosphate-binding domain and the substrate-binding domain and in dimer stabilization with the eukaryotic ODCs, were also conserved in S. ruminantium LDC. Computer analysis of the putative secondary structure of S. ruminantium LDC showed that it is approximately 70% identical to that of mouse ODC. We identified five amino acid residues, A44, G45, V46, P54, and S322, within the LDC catalytic domain that confer decarboxylase activities toward both L-lysine and L-ornithine with a substrate specificity ratio of 0.83 (defined as the k(cat)/K(m) ratio obtained with L-ornithine relative to that obtained with L-lysine). We have succeeded in converting S. ruminantium LDC to form with a substrate specificity ratio of 58 (70 times that of wild-type LDC) by constructing a mutant protein, A44V/G45T/V46P/P54D/S322A. In this study, we also showed that G350 is a crucial residue for stabilization of the dimer in S. ruminantium LDC.     

Physiological evidence for an interaction between helix XI and helices I, II, and V in the melibiose carrier of Escherichia coli

In a previous study 23 residues in helix XI of the cysteine-less melibiose carrier were changed individually to cysteine. Several of these cysteine mutants (K377C, A383C, F385C, L391C, G395C) had low transport activity and they were white on melibiose MacConkey fermentation plates. After several days of incubation of these white clones on melibiose MacConkey plates a rare red mutant appeared. The plasmid DNA was then isolated and sequenced. The two second site revertants from K377C were I22S and D59A. This change of aspartic acid to a neutral residue suggests that physiologically there is an interaction between K377 and D59 (possibly a salt bridge). The revertants from A383C were in positions 20 (F20L) and 22 (I22S and I22N). Revertants of F385C were intrahelical changes (I387M and A388G) and a change in C-terminal loop (R441C). Revertants of L391C were in helix I (I22N, I22T and D19E) and helix V (A152S). Revertants of G395C were in helix I (D19E and I22N). We suggest that there is an interaction between helix XI and helices I, II, and V and proximity between these helices.     

Drug resistance mutations can effect dimer stability of HIV-1 protease at neutral pH.

The monomer-dimer equilibrium for the human immunodeficiency virus type 1 (HIV-1) protease has been investigated under physiological conditions. Dimer dissociation at pH 7.0 was correlated with a loss in beta-sheet structure and a lower degree of ANS binding. An autolysis-resistant mutant, Q7K/L33I/L63I, was used to facilitate sedimentation equilibrium studies at neutral pH where the wild-type enzyme is typically unstable in the absence of bound inhibitor. The dimer dissociation constant (KD) of the triple mutant was 5.8 microM at pH 7.0 and was below the limit of measurement (approximately 100 nM) at pH 4.5. Similar studies using the catalytically inactive D25N mutant yielded a KD value of 1.0 microM at pH 7.0. These values differ significantly from a previously reported value of 23 nM obtained indirectly from inhibitor binding measurements (Darke et al., 1994). We show that the discrepancy may result from the thermodynamic linkage between the monomer-dimer and inhibitor binding equilibria. Under conditions where a significant degree of monomer is present, both substrates and competitive inhibitors will shift the equilibrium toward the dimer, resulting in apparent increases in dimer stability and decreases in ligand binding affinity. Sedimentation equilibrium studies were also carried out on several drug-resistant HIV-1 protease mutants: V82F, V82F/I84V, V82T/I84V, and L90M. All four mutants exhibited reduced dimer stability relative to the autolysis-resistant mutant at pH 7.0. Our results indicate that reductions in drug affinity may be due to the combined effects of mutations on both dimer stability and inhibitor binding.     

A rational approach to Re-engineer cytochrome P450 2B1 regioselectivity based on the crystal structure of cytochrome P450 2C5

The regioselectivity for progesterone hydroxylation by cytochrome P450 2B1 was re-engineered based on the x-ray crystal structure of cytochrome P450 2C5. 2B1 is a high K(m) progesterone 16alpha-hydroxylase, whereas 2C5 is a low K(m) progesterone 21-hydroxylase. Initially, nine individual 2B1 active-site residues were changed to the corresponding 2C5 residues, and the mutants were purified from an Escherichia coli expression system and assayed for progesterone hydroxylation. At 150 microm progesterone, I114A, F297G, and V363L showed 5-15% of the 21-hydroxylase activity of 2C5, whereas F206V showed high activity for an unknown product and a 13-fold decrease in K(m). Therefore, a quadruple mutant, I114A/F206V/F297G/V363L (Q), was constructed that showed 60% of 2C5 progesterone 21-hydroxylase activity and 57% regioselectivity. Based on their 2C5-like testosterone hydroxylation profiles, S294D and I477F alone and in combination were added to the quadruple mutant. All three mutants showed enhanced regioselectivity (70%) for progesterone 21-hydroxylation, whereas only Q/I477F had a higher k(cat). Finally, the remaining three single mutants, V103I, V367L, and G478V, were added to Q/I477F and Q/S294D/I477F, yielding seven additional multiple mutants. Among these, Q/V103I/S294D/I477F showed the highest k(cat) (3-fold higher than that of 2C5) and 80% regioselectivity for progesterone 21-hydroxylation. Docking of progesterone into a three-dimensional model of this mutant indicated that 21-hydroxylation is favored. In conclusion, a systematic approach to convert P450 regioselectivity across subfamilies suggests that active-site residues are mainly responsible for regioselectivity differences between 2B1 and 2C5 and validates the reliability of 2B1 models based on the crystal structure of 2C5.     

The complete amino acid sequence of coagulogen isolated from Southeast Asian horseshoe crab, Carcinoscorpius rotundicauda

The complete amino acid sequence of coagulogen purified from the hemocytes of the horseshoe crab Carcinoscorpius rotundicauda was determined by characterization of the NH2-terminal sequence and the peptides generated after digestion of the protein with lysyl endopeptidase, Staphylococcal aureus protease V8 and trypsin. Upon sequencing the peptides by the automated Edman method, the following sequence was obtained: A D T N A P L C L C D E P G I L G R N Q L V T P E V K E K I E K A V E A V A E E S G V S G R G F S L F S H H P V F R E C G K Y E C R T V R P E H T R C Y N F P P F V H F T S E C P V S T R D C E P V F G Y T V A G E F R V I V Q A P R A G F R Q C V W Q H K C R Y G S N N C G F S G R C T Q Q R S V V R L V T Y N L E K D G F L C E S F R T C C G C P C R N Y Carcinoscorpius coagulogen consists of a single polypeptide chain with a total of 175 amino acid residues and a calculated molecular weight of 19,675. The secondary structure calculated by the method of Chou and Fasman reveals the presence of an alpha-helix region in the peptide C segment (residue Nos. 19 to 46), which is released during the proteolytic conversion of coagulogen to coagulin gel. The beta-sheet structure and the 16 half-cystines found in the molecule appear to yield a compact protein stable to acid and heat. The amino acid sequences of coagulogen of four species of limulus have been compared and the interspecies evolutionary differences are discussed.     

Frequency of MEFV gene mutations in Hatay province,Mediterranean region of Turkey and report of a novel missense mutation (I247V)

In the present study, 1000 patients with clinical suspicion of FMF were retrospectively reviewed to determine the spectrum of MEFV gene mutations by using DNA sequence analysis between September, 2008 and April, 2012. Sixteen different mutations and 55 different genotypes were detected in 618 of 1000 patients. Among 16 different mutations, R202Q (21.35%) was the most frequently observed mutation; followed by E148Q (8.85%), M694V (7.95%), M680I (2.40%), V726A (1.85%), M694I (0.95%), A744S (0.80%), R761H (0.55%), P283L (0.35%), K695R (0.20%), E230K (0.15%), L110P (0.10%), I247V (0.05%), G196W (0.05%) and G304R (0.05%). In the present study, a novel missense mutation (I247V) and a silent variant (G150G) were identified in the MEFV gene. On the other hand, P238L, G632A and G304R mutations are the first cases reported from Turkey. Our results indicated that MEFV mutations are highly heterogeneous in our study population as in other regions of Turkey and mutation screening techniques such as PCR-RFLP, amplification refractory mutation system or reverse hybridization do not adequately detect uncommon or novel mutations. Therefore, it was proven that sequence analysis of the MEFV gene could be useful for detection of rare or unknown mutations.     

Construction and characterization of beta-lactoglobulin chimeras

At neutral pH, equine beta-lactoglobulin (ELG) is monomeric, whereas bovine beta-lactoglobulin (BLG) exists as a dimer. To understand the difference in the oligomerization properties between ELG and BLG, three mutants of ELG (LP, I, and LPI) were constructed by substituting amino acids responsible for important interactions at the dimer interface of BLG into ELG. The mutant LP has an AB loop mutation (S34A/E35Q), the mutant I has an I strand mutation (G145M/R146H/V147I/Q148R/I149L/V150S/P151F/D152N/L153P) and the mutant LPI includes both the LP and I mutations. The far- and near-UV CD spectra of the three mutants are similar to that of the wild-type ELG, indicating that the secondary and the tertiary structures of ELG are not significantly affected by the mutations. Ultracentrifuge analysis shows that all three mutants are monomeric at neutral pH, suggesting that the protein sequences in the AB loop and I strand of BLG alone cannot support dimerization of ELG. Thus, structural differences must exist between ELG and BLG that prevent the ELG mutants from forming the same interactions as BLG at the dimer interface.     

Importance of individual amino acids in the Switch I region in eEF2 studied by functional complementation in S. cerevisiae

Elongation factor 2 (eEF2) is a member of the G-protein super family. G-proteins undergo conformational changes associated with binding of the guanosine nucleotide and hydrolysis of the bound GTP. These structural rearrangements affects the Switch I region (also known as the Effector loop). We have studied the role of individual amino acids in the Switch I region (amino acids 25-73) of S. cerevisiae eEF2 using functional complementation in yeast. 21 point mutations in the Switch I region were created by site-directed mutagenesis. Mutants K49R, E52Q, A53G, F55Y, K60R, Q63A, T68S, I69M and A73G were functional while mutants R54H, F55N, D57A, D57E, D57S, R59K, R59M, Q63E, R65A, R65N, T68A and T68M were inactive. Expression of mutants K49R, A53G, Q63A, I69M and A73G was associated with markedly decreased growth rates and yeast cells expressing mutants A53G and I69M became temperature sensitive. The functional capacity of eEF2 in which the major part Switch I (amino acids T56 to I69) was converted into the homologous sequence found in EF-G from E. coli was also studied. This protein chimera could functionally replace yeast eEF2 in vivo. Yeast cells expressing this mutant grew extremely slowly, showed increased cell death and became temperature sensitive. The ability of the mutant to replace authentic eEF2 in vivo indicates that the structural rearrangement of Switch I necessary for eEF2 function is similar in eukaryotes and bacteria. The effect of two point mutations in the P-loop was also studied. Mutant A25G but not A25V could functionally replace yeast eEF2 even if cells expressing the mutant grew slowly. The A25G mutation converted the consensus sequences AXXXXGK[T/S] in eEF2 to the corresponding motif GXXXXGK[T/S] found in all other G-proteins, suggesting that the alanine found in the P-loop of peptidyltranslocases are not essential for function.