Specificity and cooperativity at β-lactamase position 104 in TEM-1/BLIP and SHV-1/BLIP interactions

Establishing a quantitative understanding of the determinants of affinity in protein–protein interactions remains challenging. For example, TEM‐1/β‐lactamase inhibitor protein (BLIP) and SHV‐1/BLIP are homologous β‐lactamase/β‐lactamase inhibitor protein complexes with disparate K_d values (3 nM and 2 μM, respectively), and a single substitution, D104E in SHV‐1, results in a 1000‐fold enhancement in binding affinity. In TEM‐1, E104 participates in a salt bridge with BLIP K74, whereas the corresponding SHV‐1 D104 does not in the wild type SHV‐1/BLIP co‐structure. Here, we present a 1.6 Å crystal structure of the SHV‐1 D104E/BLIP complex that demonstrates that this point mutation restores this salt bridge. Additionally, mutation of a neighboring residue, BLIP E73M, results in salt bridge formation between SHV‐1 D104 and BLIP K74 and a 400‐fold increase in binding affinity. To understand how this salt bridge contributes to complex affinity, the cooperativity between the E/K or D/K salt bridge pair and a neighboring hot spot residue (BLIP F142) was investigated using double mutant cycle analyses in the background of the E73M mutation. We find that BLIP F142 cooperatively stabilizes both interactions, illustrating how a single mutation at a hot spot position can drive large perturbations in interface stability and specificity through a cooperative interaction network. Proteins 2011. © 2011 Wiley‐Liss, Inc.     

Differential effects on enzyme stability and kinetic parameters of mutants related to human triosephosphate isomerase deficiency

Human triosephosphate isomerase (TIM) deficiency is a very rare disease, but there are several mutations reported to be causing the illness. In this work, we produced nine recombinant human triosephosphate isomerases which have the mutations reported to produce TIM deficiency. These enzymes were characterized biophysically and biochemically to determine their kinetic and stability parameters, and also to substitute TIM activity in supporting the growth of an Escherichia coli strain lacking the tim gene. Our results allowed us to rate the deleteriousness of the human TIM mutants based on the type and severity of the alterations observed, to classify four “unknown severity mutants” with altered residues in positions 62, 72, 122 and 154 and to explain in structural terms the mutation V231M, the most affected mutant from the kinetic point of view and the only homozygous mutation reported besides E104D.     

Ramachandran analysis of conserved glycyl residues in homologous proteins of known structure

High conservation of glycyl residues in homologous proteins is fairly frequent. It is commonly understood that glycine tends to be highly conserved either because of its unique Ramachandran angles or to avoid steric clash that would arise with a larger side chain. Using a database of aligned 3D structures of homologous proteins we identified conserved Gly in 288 alignment positions from 85 families. Ninety‐six of these alignment positions correspond to conserved Gly residue with (φ, ψ) values allowed for non‐glycyl residues. Reasons for this observation were investigated by in‐silico mutation of these glycyl residues to Ala. We found in 94% of the cases a short contact exists between the C^β atom of the introduced Ala with the atoms which are often distant in the primary structure. This suggests the lack of space even for a short side chain thereby explaining high conservation of glycyl residues even when they adopt (φ, ψ) values allowed for Ala. In 189 alignment positions, the conserved glycyl residues adopt (φ, ψ) values which are disallowed for Ala. In‐silico mutation of these Gly residues to Ala almost always results in steric hindrance involving C^β atom of Ala as one would expect by comparing Ramachandran maps for Ala and Gly. Rare occurrence of the disallowed glycyl conformations even in ultrahigh resolution protein structures are accompanied by short contacts in the crystal structures and such disallowed conformations are not conserved in the homologues. These observations raise the doubt on the accuracy of such glycyl conformations in proteins.     

Hydrophobic effect on the stability and folding of a hyperthermophilic protein

Ribonuclease HII from hyperthermophile Thermococcus kodakaraensis (Tk-RNase HII) is a kinetically robust monomeric protein. The conformational stability and folding kinetics of Tk-RNase HII were measured for nine mutant proteins in which a buried larger hydrophobic side chain is replaced by a smaller one (Leu/Ile to Ala). The mutant proteins were destabilized by 8.9 to 22.0 kJ mol^− 1 as compared with the wild-type protein. The removal of each -CH₂- group burial decreased the stability by 5.1 kJ mol^− 1 on average in the mutant proteins of Tk-RNase HII examined. This is comparable with the value of 5.3 kJ mol^− 1 obtained from experiments for proteins from organisms growing at moderate temperature. We conclude that the hydrophobic residues buried inside protein molecules contribute to the stabilization of hyperthermophilic proteins to a similar extent as proteins at normal temperature. In the folding experiments, the mutant proteins of Tk-RNase HII examined exhibited faster unfolding compared with the wild-type protein. These results indicate that the buried hydrophobic residues strongly contribute to the kinetic robustness of Tk-RNase HII. This is the first report that provides a practical cause of slow unfolding of hyperthermostable proteins.     

Evidence for a Micrococcus luteus gene homologous to uvrB of Escherichia coli

Summary Restriction fragments of Micrococcus luteus DNA that contained the gene defined by the mutation of an excision repair-deficient mutant, UV^sN1, were cloned from both the parental and mutant strains with the Escherichia coli host-vector system. The wild-type fragment was able to reverse the multiple sensitivity of the mutant to ultraviolet, mitomycin C, and 4-nitroquinoline-1-oxide by one-step transformation. Determination of the nucleotide sequences revealed an open reading frame potentially coding for a protein of 709 amino acid residues, within which the mutation was identified as a CGTA transition causing a change from serine to phenylalanine. The putative product of the open reading frame showed an extensive amino acid sequence homology to the E. coli UvrB protein comprising 673 residues; the homologous region extended over the greater parts of both polypeptides, in which 55% and 17% of the 659 pairs of aligned amino acids were accounted for by conserved residues and conservative substitutions, respectively. This indicates that the gene defined by the UV^sN1 mutation represents a homolog of the E. coli uvrB gene, implying the presence in M. luteus of an enzyme complex homologous to the E. coli UvrABC excinuclease.Abbreviations Ap ampicillin - AP apurinic-apyrimidinic - MC mitomycin - C: 4NQO 4-nitroquinoline-1-oxide - r resistant - s sensitive - UV ultraviolet Dedicated to the memory of Shunzo Okubo (1930–1978) who played the pivotal role in our earlier studies on the M. luteus repair system     

The critical role of the loops of triosephosphate isomerase for its oligomerization,dynamics, and functionality

Triosephosphate isomerase (TIM) catalyzes the reaction to convert dihydroxyacetone phosphate into glyceraldehyde 3‐phosphate, and vice versa. In most organisms, its functional oligomeric state is a homodimer; however, tetramer formation in hyperthermophiles is required for functional activity. The tetrameric TIM structure also provides added stability to the structure, enabling it to function at more extreme temperatures. We apply Principal Component Analysis to find that the TIM structure space is clearly divided into two groups—the open and the closed TIM structures. The distribution of the structures in the open set is much sparser than that in the closed set, showing a greater conformational diversity of the open structures. We also apply the Elastic Network Model to four different TIM structures—an engineered monomeric structure, a dimeric structure from a mesophile—Trypanosoma brucei, and two tetrameric structures from hyperthermophiles Thermotoga maritima and Pyrococcus woesei. We find that dimerization not only stabilizes the structures, it also enhances their functional dynamics. Moreover, tetramerization of the hyperthermophilic structures increases their functional loop dynamics, enabling them to function in the destabilizing environment of extreme temperatures. Computations also show that the functional loop motions, especially loops 6 and 7, are highly coordinated. In summary, our computations reveal the underlying mechanism of the allosteric regulation of the functional loops of the TIM structures, and show that tetramerization of the structure as found in the hyperthermophilic organisms is required to maintain the coordination of the functional loops at a level similar to that in the dimeric mesophilic structure.     

Metabolism in 1,3‐propanediol fed‐batch fermentation by a D‐lactate deficient mutant of Klebsiella pneumoniae

Klebsiella pneumoniae HR526, a new isolated 1,3‐propanediol (1,3‐PD) producer, exhibited great productivity. However, the accumulation of lactate in the late‐exponential phase remained an obstacle of 1,3‐PD industrial scale production. Hereby, mutants lacking D ‐lactate pathway were constructed by knocking out the ldhA gene encoding fermentative D ‐lactate dehydrogenase (LDH) of HR526. The mutant K. pneumoniae LDH526 with the lowest LDH activity was studied in aerobic fed‐batch fermentation. In experiments using pure glycerol as feedstock, the 1,3‐PD concentrations, conversion, and productivity increased from 95.39 g L^?1, 0.48 and 1.98 g L^?1 h^?1 to 102. 06 g L^?1, 0.52 mol mol^?1 and 2.13 g L^?1 h^?1, respectively. The diol (1,3‐PD and 2,3‐butanediol) conversion increased from 0.55 mol mol^?1 to a maximum of 0.65 mol mol^?1. Lactate would not accumulate until 1,3‐PD exceeded 84 g L^?1, and the final lactate concentration decreased dramatically from more than 40 g L^?1 to <3 g L^?1. Enzymic measurements showed LDH activity decreased by 89–98% during fed‐batch fermentation, and other related enzyme activities were not affected. NADH/NAD⁺ enhanced more than 50% in the late‐exponential phase as the D ‐lactate pathway was cut off, which might be the main reason for the change of final metabolites concentrations. The ability to utilize crude glycerol from biodiesel process and great genetic stability demonstrated that K. pnemoniae LDH526 was valuable for 1,3‐PD industrial production. Biotechnol. Bioeng. 2009; 104: 965–972. © 2009 Wiley Periodicals, Inc.     

Phylogenetic analysis of eIF4E-family members

Background  

Translation initiation in eukaryotes involves the recruitment of mRNA to the ribosome which is controlled by the translation factor eIF4E. eIF4E binds to the 5'-m⁷Gppp cap-structure of mRNA. Three dimensional structures of eIF4Es bound to cap-analogues resemble 'cupped-hands' in which the cap-structure is sandwiched between two conserved Trp residues (Trp-56 and Trp-102 of H. sapiens eIF4E). A third conserved Trp residue (Trp-166 of H. sapiens eIF4E) recognizes the⁷-methyl moiety of the cap-structure. Assessment of GenBank NR and dbEST databases reveals that many organisms encode a number of proteins with homology to eIF4E. Little is understood about the relationships of these structurally related proteins to each other.     

Conformational stability of α-amylase from malted sorghum (Sorghum bicolor): Reversible unfolding by denaturants

α-Amylase from Sorghum bicolor, is reversibly unfolded by chemical denaturants at pH 7.0 in 50 mM Hepes containing 13.6 mM calcium and 15 mM DTT. The isothermal equilibrium unfolding at 27 °C is characterized by two state transition with ΔG (H₂O) of 16.5 kJ mol⁻¹ and 22 kJ mol⁻¹, respectively, at pH 4.8 and pH 7.0 for GuHCl and ΔG (H₂O) of 25.2 kJ mol⁻¹ at pH 4.8 for urea. The conformational stability indicators such as the change in excess heat capacity (ΔC_p), the unfolding enthalpy (H_g) and the temperature at ΔG = 0 (T_g) are 17.9 ± 0.7 kJ mol⁻¹ K⁻¹, 501.2 ± 18.2 kJ mol⁻¹ and 337.3 ± 6.9 K at pH 4.8 and 14.3 ± 0.5 kJ mol⁻¹ K⁻¹, 509.3 ± 21.7 kJ mol⁻¹ and 345.4 ± 4.8 K at pH 7.0, respectively. The reactivity of the conserved cysteine residues, during unfolding, indicates that unfolding starts from the ‘B’ domain of the enzyme. The oxidation of cysteine residues, during unfolding, can be prevented by the addition of DTT. The conserved cysteine residues are essential for enzyme activity but not for the secondary and tertiary fold acquired during refolding of the denatured enzyme. The pH dependent stability described by ΔG (H₂O) and the effect of salt on urea induced unfolding confirm the role of electrostatic interactions in enzyme stability.     

Structural basis of human triosephosphate isomerase deficiency: mutation E104D is related to alterations of a conserved water network at the dimer interface

Human triosephosphate isomerase deficiency is a rare autosomal disease that causes premature death of homozygous individuals. The most frequent mutation that leads to this illness is in position 104, which involves a conservative change of a Glu for Asp. Despite the extensive work that has been carried out on the E104D mutant enzyme in hemolysates and whole cells, the molecular basis of this disease is poorly understood. Here, we show that the purified, recombinant mutant enzyme E104D, while exhibiting normal catalytic activity, shows impairments in the formation of active dimers and low thermostability and monomerizes under conditions in which the wild type retains its dimeric form. The crystal structure of the E104D mutant at 1.85 A resolution showed that its global structure was similar to that of the wild type; however, residue 104 is part of a conserved cluster of 10 residues, five from each subunit. An analysis of the available high resolution structures of TIM dimers revealed that this cluster forms a cavity that possesses an elaborate conserved network of buried water molecules that bridge the two subunits. In the E104D mutant, a disruption of contacts of the amino acid side chains in the conserved cluster leads to a perturbation of the water network in which the water-protein and water-water interactions that join the two monomers are significantly weakened and diminished. Thus, the disruption of this solvent system would stand as the underlying cause of the deficiency.     

Mutations inside but not outside the peptide binding cleft of the H-2 Ld molecule affects CTL recognition and binding of the nucleoprotein peptide from the lymphocytic chroriomeningitis

In order to investigate the role of residues inside and outside the peptide binding cleft of the L² molecule in peptide presentation to cytotoxic T lymphocytes (CTL), we constructed a series of point mutations in the L ^d gene. We determined the effects of the mutations in the L^d molecule on the binding and recognition of an L^d-restricted CTL epitope derived from the nucleoprotein (NP) of the lymphocytic phoriomeningitis virus (LCMV). Each of the mutations within the L^d peptide binding cleft resulted in a complete loss of CTL recognition. Addition of the LCMV NP peptide to cells expressing these mutants did not increase surface L^d expression, suggesting that the mutations altered peptide binding. Mutations involving pockets D and E within the cleft affected LCMV peptide binding and recognition as drastically as those in pocket B, which was predicted to interact with a main anchor residue of the peptide. In striking contrast, the mutations located outside the cleft did not change either recognition or binding. These results demonstrate that the L^d residues in the peptide binding cleft are the main determinants dictating LCMV NP peptide binding, and that the residues in each of the pockets within the cleft play a role in this interaction. Surprisingly, one mutation outside the peptide binding cleft, T92S, abrogated CTL lysis of target cells treated with the LCMV NP peptide, but not virus-infected cells. These data show that this mutation selectively altered the presentation of the LCMV NP peptide introduced to the cell exogenously, but not endogenously. This implies that the pathway by which peptides associate with class I molecules within the cell differs from that of exogenous peptide binding.     

RNase E forms a complex with polynucleotide phosphorylase in cyanobacteria via a cyanobacterial-specific nonapeptide in the noncatalytic region

RNase E, a central component involved in bacterial RNA metabolism, usually has a highly conserved N-terminal catalytic domain but an extremely divergent C-terminal domain. While the C-terminal domain of RNase E in Escherichia coli recruits other components to form an RNA degradation complex, it is unknown if a similar function can be found for RNase E in other organisms due to the divergent feature of this domain. Here, we provide evidence showing that RNase E forms a complex with another essential ribonuclease—the polynucleotide phosphorylase (PNPase)—in cyanobacteria, a group of ecologically important and phylogenetically ancient organisms. Sequence alignment for all cyanobacterial RNase E proteins revealed several conserved and variable subregions in their noncatalytic domains. One such subregion, an extremely conserved nonapeptide (RRRRRRSSA) located near the very end of RNase E, serves as the PNPase recognition site in both the filamentous cyanobacterium Anabaena PCC7120 and the unicellular cyanobacterium Synechocystis PCC6803. These results indicate that RNase E and PNPase form a ribonuclease complex via a common mechanism in cyanobacteria. The PNPase-recognition motif in cyanobacterial RNase E is distinct from those previously identified in Proteobacteria, implying a mechanism of coevolution for PNPase and RNase E in different organisms.     

Kinetics and thermodynamics of catalysis and thermal inactivation of a novel α-amylase (Tp-AmyS) from Thermotoga petrophila

Abstract

This research is focussed on kinetic, thermodynamic and thermal inactivation of a novel thermostable recombinant α-amylase (Tp-AmyS) from Thermotoga petrophila. The amylase gene was cloned in pHIS-parallel1 expression vector and overexpressed in Escherichia coli. The steady-state kinetic parameters (V_max, K_m, k_cat and k_cat/K_m) for the hydrolysis of amylose (1.39?mg/min, 0.57?mg, 148.6?s^?1, 260.7), amylopectin (2.3?mg/min, 1.09?mg, 247.1?s^?1, 226.7), soluble starch (2.67?mg/min, 2.98?mg, 284.2?s^?1, 95.4) and raw starch (2.1?mg/min, 3.6?mg, 224.7?s^?1, 61.9) were determined. The activation energy (E_a), free energy (ΔG), enthalpy (ΔH) and entropy of activation (ΔS) at 98?°C were 42.9?kJ mol^?1, 74?kJ mol^?1, 39.9?kJ mol^?1 and ?92.3 J mol^?1 K^?1, respectively, for soluble starch hydrolysis. While ΔG of substrate binding (ΔG_E-S) and ΔG of transition state binding (ΔG_E-T) were 3.38 and ?14.1?kJ mol^?1, respectively. Whereas, EaD, Gibbs free energy (ΔG*), increase in the enthalpy (ΔH*) and activation entropy (ΔS*) for activation of the unfolding of transition state were 108, 107, 105?kJ mol^?1 and ?4.1 J mol^?1 K^?1. The thermodynamics of irreversible thermal inactivation of Tp-AmyS revealed that at high temperature the process involves the aggregation of the protein.     

A Conserved Salt Bridge between Transmembrane Segments 1 and 10 Constitutes an Extracellular Gate in the Dopamine Transporter

Neurotransmitter transporters play an important role in termination of synaptic transmission by mediating reuptake of neurotransmitter, but the molecular processes behind translocation are still unclear. The crystal structures of the bacterial homologue, LeuT, provided valuable insight into the structural and dynamic requirements for substrate transport. These structures support the existence of gating domains controlling access to a central binding site. On the extracellular side, access is controlled by the “thin gate” formed by an interaction between Arg-30 and Asp-404. In the human dopamine transporter (DAT), the corresponding residues are Arg-85 and Asp-476. Here, we present results supporting the existence of a similar interaction in DAT. The DAT R85D mutant has a complete loss of function, but the additional insertion of an arginine in opposite position (R85D/D476R), causing a charge reversal, results in a rescue of binding sites for the cocaine analogue [³H]CFT. Also, the coordination of Zn²⁺ between introduced histidines (R85H/D476H) caused a ∼2.5-fold increase in [³H]CFT binding (B_max). Importantly, Zn²⁺ also inhibited [³H]dopamine transport in R85H/D476H, suggesting that a dynamic interaction is required for the transport process. Furthermore, cysteine-reactive chemistry shows that mutation of the gating residues causes a higher proportion of transporters to reside in the outward facing conformation. Finally, we show that charge reversal of the corresponding residues (R104E/E493R) in the serotonin transporter also rescues [³H](S)-citalopram binding, suggesting a conserved feature. Taken together, these data suggest that the extracellular thin gate is present in monoamine transporters and that a dynamic interaction is required for substrate transport.     

βα-Hairpin Clamps Brace βαβ Modules and Can Make Substantive Contributions to the Stability of TIM Barrel Proteins

Non-local hydrogen bonding interactions between main chain amide hydrogen atoms and polar side chain acceptors that bracket consecutive βα or αβ elements of secondary structure in αTS from E. coli, a TIM barrel protein, have previously been found to contribute 4–6 kcal mol⁻¹ to the stability of the native conformation. Experimental analysis of similar βα-hairpin clamps in a homologous pair of TIM barrel proteins of low sequence identity, IGPS from S. solfataricus and E. coli, reveals that this dramatic enhancement of stability is not unique to αTS. A survey of 71 TIM barrel proteins demonstrates a 4-fold symmetry for the placement of βα-hairpin clamps, bracing the fundamental βαβ building block and defining its register in the (βα)₈ motif. The preferred sequences and locations of βα-hairpin clamps will enhance structure prediction algorithms and provide a strategy for engineering stability in TIM barrel proteins.     

Determination of glucose-fructose oxidoreductase activity in whole cells of Zymomonas mobilis

A method for the determination of glucose-fructose oxidoreductase (GFOR) activity in whole cells of Zymomonas mobilis is described. The K _m and the theoretical V _max for GFOR were 192 g glucose.l^-1 and 17 g gluconic acid.g^-1 cell.h^-1, respectively. The changes in enthalpy (31.1 kJ.mol^-1), entropy (0.41 kJ.K^-1), and Gibbs free energy (-97.5 kJ.mol^-1) related to glucose to gluconic acid conversion were also determined.     

The conformation and aggregation of bovine β-casein A. II. Thermodynamics of thermal association and the effects of changes in polar and apolar interactions on micellization

A sedimentation analysis has been used to determine the proportion of protein present as monomer and aggregate in 0.5 and 1.0 g/dl solutions of β-casein A in pH 7 phosphate buffer over the temperature range 10–40°C. The amount and molecular weight of the aggregate increase with temperature; under the conditions used, the aggregation number (n) of β-casein is given approximately by n = 0.6t + 2 with t in degrees centigrade. The concentration of β-casein in monomeric and aggregated states at different temperatures is used to calculate the standard enthalpy of aggregation ΔH° (Van't Hoff) by assuming that β-casein undergoes a cooperative, two-state, micellization process; aggregation is an endothermic process and ΔH° = 66.0 ± 2.6 kJ mol^?1. Combination of this ΔH° with the amount of protein calculated to dissociate when 1 g/dl solutions are diluted isothermally to 0.5 g/dl gives the heat of dilution at various temperatures. These calculated heats of dilution are compared with the experimental values obtained by carrying out the same dilutions in a microcalorimeter. The heat of dilution decreases linearly with β-casein concentration, but the extrapolated zero-concentration values of 65.8 ± 1.6 kJ mol^?1 is the same as the Van't Hoff enthalpy. This agreement in the enthalpy values indicates that the micellization of β-casein occurs cooperatively. The effect of modifying the hydrophobic/hydrophilic balance of the system on the micellization of β-casein A has been investigated. The hydrophobic interaction between the protein molecules is decreased by removing the three C-terminal residues (Ileu Ileu Val) with carboxypeptidase-A. This modification drastically reduces the ability of the β-casein molecule to form micelles. Substitution of ²H₂O for H₂O at constant temperature perturbs the monomer–micelle equilibrium in favor of micelles because of enhanced hydrophobic interactions in the former solvent. The results are consistent with β-casein micellization involving a delicate balance of the hydrophobic forces favoring aggregation and electrostatic forces opposing it.     

Ecophysiological studies on citrate-synthase: (I) enzyme regulation of selected crustaceans with regard to temperature adaptation

The characteristics and properties chromatographically purified citrate synthase from the euphausiids Euphausia superba (Antarctica) and Meganyctiphanes norvegica (Scandinavian Kattegat and Mediterranean Sea) and from the isopods Serolis polita (Antarctica) and Idotea baltica (Baltic Sea) were used to elucidate biochemical mechanisms of temperature adaptation. Additionally, maintenance experiments were carried out on the euphausiids to determine mechanisms of short term acclimation. Temperature optima (between 37 and 45°C) were unrelated to genotypic cold adaptation, but the activation energy of the Antarctic krill E. superba (10.9 kJ · mol^-1) was only a quarter of that in other species (41.8–45.1 kJ · mol^-1). The minima of apparent Michaelis constants (total range: 4–20 μmol · 1^-1 oxaloacetate; 7–45 μmol · 1^-1 acetyl-coenzyme A) showed no relation to natural conditions, and no distinct pH optimum occurred at ambient temperatures. In contrast, apparent Michaelis constants and specific enzyme activities were related to maintenance temperatures in M. norvegica, but not in E. superba. The differences between M. norvegica and E. superba can be interpreted as adaptations to the changes in ambient temperature with regard to the respective steno- and eurythermic tolerances of these crustaceans.