Thermodynamic investigation of the role of contact residues of beta-lactamase-inhibitory protein for binding to TEM-1 beta-lactamase

We have determined the thermodynamics of binding for the interaction between TEM-1 beta-lactamase and a set of alanine substituted contact residue mutants ofbeta-lactamase-inhibitory protein (BLIP) using isothermal titration calorimetry. The binding enthalpies for these interactions are highly temperature dependent, with negative binding heat capacity changes ranging from -800 to -271 cal mol(-1) K(-1). The isoenthalpic temperatures (at which the binding enthalpy is zero) of these interactions range from 5 to 38 degrees C. The changes in isoenthalpic temperature were used as an indicator of the changes in enthalpy and entropy driving forces, which in turn are related to hydrophobic and hydrophilic interactions. A contact residue of BLIP is categorized as a canonical residue if its alanine substitution mutant exhibits a change of isoenthalpic temperature matching the change of hydrophobicity because of the mutation. A contact position exhibiting a change in isoenthalpic temperature that does not match the change in hydrophobicity is categorized as an anti-canonical residue. Our experimental results reveal that the majority of residues where alanine substitution results in a loss of affinity are canonical (7 of 10), and about half of the residues where alanine substitutions have a minor effect are canonical. The interactions between TEM-1beta-lactamase and BLIP canonical contact residues contribute directly to binding free energy, suggesting potential anchoring sites for binding partners. The anti-canonical behavior of certain residues may be the result of mutation-induced modifications such as structural rearrangements affecting contact residue configurations. Structural inspection of BLIP suggests that the Lys(74) side chain electrostatically holds BLIP loop 2 in position to bind to TEM-1 beta-lactamase, explaining a large loss of entropy-driven binding energy of the K74A mutant and the resulting anti-canonical behavior. The anti-canonical behavior of the W150A mutant may also be due to structural rearrangements. Finally, the affinity enhancing effect of the contact residue mutant Y50A may be due to energetic coupling interactions between Asp(49) and His(41).     

Design of potent beta-lactamase inhibitors by phage display of beta-lactamase inhibitory protein

Beta-lactamase inhibitory protein (BLIP) binds tightly to several beta-lactamases including TEM-1 beta-lactamase (K(i) 0.1 nm). The TEM-1 beta-lactamase/BLIP co-crystal structure indicates that two turn regions in BLIP insert into the active site of beta-lactamase to block the binding of beta-lactam antibiotics. Residues from each turn, Asp(49) and Phe(142), mimic interactions made by penicillin G when bound in the beta-lactamase active site. Phage display was used to determine which residues within the turn regions of BLIP are critical for binding TEM-1 beta-lactamase. The sequences of a set of functional mutants from each library indicated that a few sequence types were predominant. These BLIP mutants exhibited K(i) values for beta-lactamase inhibition ranging from 0.01 to 0.2 nm. The results indicate that even though BLIP is a potent inhibitor of TEM-1 beta-lactamase, the wild-type sequence of the active site binding region is not optimal and that derivatives of BLIP that bind beta-lactamase extremely tightly can be obtained. Importantly, all of the tight binding BLIP mutants have sequences that would be predicted theoretically to form turn structures.     

Identification of residues in beta-lactamase critical for binding beta-lactamase inhibitory protein

beta-Lactamase inhibitory protein (BLIP) is a potent inhibitor of several beta-lactamases including TEM-1 beta-lactamase (Ki = 0.1 nM). The co-crystal structure of TEM-1 beta-lactamase and BLIP has been solved, revealing the contact residues involved in the interface between the enzyme and inhibitor. To determine which residues in TEM-1 beta-lactamase are critical for binding BLIP, the method of monovalent phage display was employed. Random mutants of TEM-1 beta-lactamase in the 99-114 loop-helix and 235-240 B3 beta-strand regions were displayed as fusion proteins on the surface of the M13 bacteriophage. Functional mutants were selected based on the ability to bind BLIP. After three rounds of enrichment, the sequences of a collection of functional beta-lactamase mutants revealed a consensus sequence for the binding of BLIP. Seven loop-helix residues including Asp-101, Leu-102, Val-103, Ser-106, Pro-107, Thr-109, and His-112 and three B3 beta-strand residues including Ser-235, Gly-236, and Gly-238 were found to be critical for tight binding of BLIP. In addition, the selected beta-lactamase mutants A113L/T114R and E240K were found to increase binding of BLIP by over 6- and 11-fold, respectively. Combining these substitutions resulted in 550-fold tighter binding between the enzyme and BLIP with a Ki of 0.40 pM. These results reveal that the binding between TEM-1 beta-lactamase and BLIP can be improved and that there are a large number of sequences consistent with tight binding between BLIP and beta-lactamase.     

Structural and computational characterization of the SHV-1 beta-lactamase-beta-lactamase inhibitor protein interface

Beta-lactamase inhibitor protein (BLIP) binds a variety of class A beta-lactamases with affinities ranging from micromolar to picomolar. Whereas the TEM-1 and SHV-1 beta-lactamases are almost structurally identical, BLIP binds TEM-1 approximately 1000-fold tighter than SHV-1. Determining the underlying source of this affinity difference is important for understanding the molecular basis of beta-lactamase inhibition and mechanisms of protein-protein interface specificity and affinity. Here we present the 1.6A resolution crystal structure of SHV-1.BLIP. In addition, a point mutation was identified, SHV D104E, that increases SHV.BLIP binding affinity from micromolar to nanomolar. Comparison of the SHV-1.BLIP structure with the published TEM-1.BLIP structure suggests that the increased volume of Glu-104 stabilizes a key binding loop in the interface. Solution of the 1.8A SHV D104K.BLIP crystal structure identifies a novel conformation in which this binding loop is removed from the interface. Using these structural data, we evaluated the ability of EGAD, a program developed for computational protein design, to calculate changes in the stability of mutant beta-lactamase.BLIP complexes. Changes in binding affinity were calculated within an error of 1.6 kcal/mol of the experimental values for 112 mutations at the TEM-1.BLIP interface and within an error of 2.2 kcal/mol for 24 mutations at the SHV-1.BLIP interface. The reasonable success of EGAD in predicting changes in interface stability is a promising step toward understanding the stability of the beta-lactamase.BLIP complexes and computationally assisted design of tight binding BLIP variants.     

Dissecting the protein-protein interface between beta-lactamase inhibitory protein and class A beta-lactamases

beta-Lactamase inhibitory protein (BLIP) binds and inhibits a diverse collection of class A beta-lactamases at a wide range of affinities. Alanine-scanning mutagenesis was previously performed to identify the amino acid sequence requirements of BLIP for inhibiting TEM-1 beta-lactamase and SME-1 beta-lactamase. Two hotspots of binding energy, one from each domain of BLIP, were identified (Zhang, Z., and Palzkill, T. (2003) J. Biol. Chem. 278, 45706-45712). This study has been extended to examine the amino acid sequence requirements of BLIP for binding to the SHV-1 beta-lactamase, which is a poor binding substrate (Ki= 1.1 microm), and the Bacillus anthracis Bla1 enzyme (Ki= 2.5 nm). The two hotspots previously identified as important for binding TEM-1 and SME-1 beta-lactamase were also found to be important for binding Bla1. The hotspot from the second domain of BLIP, however, does not make substantial contributions to SHV-1 binding. This may explain why BLIP binds to SHV-1 beta-lactamase with much weaker affinity than to the other three enzymes. Three regions, including two loops that insert into the active pocket of TEM-1 beta-lactamase and the Glu-73-Lys-74 buried charge motif, exhibit strikingly different effects on the binding affinity of BLIP toward the various enzymes when mutated and, therefore, act as specificity determinants. Analysis of double mutants of BLIP that combine specificity-determining residues suggests that these residues contribute to the poor affinity between the second domain of BLIP and SHV-1 beta-lactamase.     

Determinants of binding affinity and specificity for the interaction of TEM-1 and SME-1 beta-lactamase with beta-lactamase inhibitory protein

The hydrolysis of beta-lactam antibiotics by class A beta-lactamases is a common cause of bacterial resistance to these agents. The beta-lactamase inhibitory protein (BLIP) is able to bind and inhibit several class A beta-lactamases, including TEM-1 beta-lactamase and SME-1 beta-lactamase. Although the TEM-1 and SME-1 enzymes share 33% amino acid sequence identity and a similar fold, they differ substantially in surface electrostatic properties and the conformation of a loop-helix region that BLIP binds. Alanine-scanning mutagenesis was performed to identify the residues on BLIP that contribute to its binding affinity for each of these enzymes. The results indicate that the sequence requirements for binding are similar for both enzymes with most of the binding free energy provided by two patches of aromatic residues on the surface of BLIP. Polar residues such as several serines in the interface do not make significant contributions to affinity for either enzyme. In addition, the specificity of binding is significantly altered by mutation of two charged residues, Glu73 and Lys74, that are buried in the structure of the TEM-1.BLIP complex as well as by residues located on two loops that insert into the active site pocket. Based on the results, a E73A/Y50A double mutant was constructed that exhibited a 220,000-fold change in binding specificity for the TEM-1 versus SME-1 enzymes.     

Protein minimization by random fragmentation and selection

Protein-protein interactions are involved in most biological processes and are important targets for drug design. Over the past decade, there has been increased interest in the design of small molecules that mimic functional epitopes of protein inhibitors. BLIP is a 165 amino acid protein that is a potent inhibitor of TEM-1 beta-lactamase (K(i) = 0.1 nM). To aid in the development of new inhibitors of beta-lactamase, the gene encoding BLIP was randomly fragmented and DNA segments encoding peptides that retain the ability to bind TEM-1 beta-lactamase were isolated using phage display. The selected peptides revealed a common, overlapping region that includes BLIP residues C30-D49. Synthesis and binding analysis of the C30-D49 peptide indicate that this peptide inhibits TEM-1 beta-lactamase. Therefore, a peptide derivative of BLIP that has been reduced in size by 88% compared with wild-type BLIP retains the ability to bind and inhibit beta-lactamase.     

Structure of the SHV-1 beta-lactamase

The X-ray crystallographic structure of the SHV-1 beta-lactamase has been established. The enzyme crystallizes from poly(ethylene glycol) at pH 7 in space group P212121 with cell dimensions a = 49.6 A, b = 55.6 A, and c = 87.0 A. The structure was solved by the molecular replacement method, and the model has been refined to an R-factor of 0.18 for all data in the range 8.0-1.98 A resolution. Deviations of model bonds and angles from ideal values are 0.018 A and 1.8 degrees, respectively. Overlay of all 263 alpha-carbon atoms in the SHV-1 and TEM-1 beta-lactamases results in an rms deviation of 1.4 A. Largest deviations occur in the H10 helix (residues 218-224) and in the loops between strands in the beta-sheet. All atoms in residues 70, 73, 130, 132, 166, and 234 in the catalytic site of SHV-1 deviate only 0.23 A (rms) from atoms in TEM-1. However, the width of the substrate binding cavity in SHV-1, as measured from the 104-105 and 130-132 loops on one side to the 235-238 beta-strand on the other side, is 0.7-1.2 A wider than in TEM-1. A structural analysis of the highly different affinity of SHV-1 and TEM-1 for the beta-lactamase inhibitory protein BLIP focuses on interactions involving Asp/Glu104.     

Computational redesign of the SHV-1 beta-lactamase/beta-lactamase inhibitor protein interface

β-lactamases are enzymes that catalyze the hydrolysis of β-lactam antibiotics. β-lactamase/β-lactamase inhibitor protein (BLIP) complexes are emerging as a well characterized experimental model system for studying protein-protein interactions. BLIP is a 165 amino acid protein that inhibits several class A β-lactamases with a wide range of affinities: picomolar affinity for K1; nanomolar affinity for TEM-1, SME-1, and BlaI; but only micromolar affinity for SHV-1 β-lactamase. The large differences in affinity coupled with the availability of extensive mutagenesis data and high-resolution crystal structures for the TEM-1/BLIP and SHV-1/BLIP complexes make them attractive systems for the further development of computational design methodology. We used EGAD, a physics-based computational design program, to redesign BLIP in an attempt to increase affinity for SHV-1. Characterization of several of designs and point mutants revealed that in all cases, the mutations stabilize the interface by 10- to 1000-fold relative to wild type BLIP. The calculated changes in binding affinity for the mutants were within a mean absolute error of 0.87 kcal/mol from the experimental values, and comparison of the calculated and experimental values for a set of 30 SHV-1/BLIP complexes yielded a correlation coefficient of 0.77. Structures of the two complexes with the highest affinity, SHV-1/BLIP (E73M) and SHV-1/BLIP (E73M, S130K, S146M), are presented at 1.7 Å resolution. While the predicted structures have much in common with the experimentally determined structures, they do not coincide perfectly; in particular a salt bridge between SHV-1 D104 and BLIP K74 is observed in the experimental structures, but not in the predicted design conformations. This discrepancy highlights the difficulty of modeling salt bridge interactions with a protein design algorithm that approximates side chains as discrete rotamers. Nevertheless, while local structural features of the interface were sometimes miscalculated, EGAD is globally successful in designing complexes with increased affinity.     

Crystal structure and kinetic analysis of beta-lactamase inhibitor protein-II in complex with TEM-1 beta-lactamase

The structure of the 28 kDa beta-lactamase inhibitor protein-II (BLIP-II) in complex with the TEM-1 beta-lactamase has been determined to 2.3 A resolution. BLIP-II is a secreted protein produced by the soil bacterium Streptomyces exfoliatus SMF19 and is able to bind and inhibit TEM-1 with subnanomolar affinity. BLIP-II is a seven-bladed beta-propeller with a unique blade motif consisting of only three antiparallel beta-strands. The overall fold is highly similar to the core structure of the human regulator of chromosome condensation (RCC1). Although BLIP-II does not share the same fold with BLIP, the first beta-lactamase inhibitor protein for which structural data was available, a comparison of the two complexes reveals a number of similarities and provides further insights into key components of the TEM-1-BLIP and TEM-1-BLIP-II interfaces. Our preliminary results from gene knock-out studies and scanning electron microscopy also reveal a critical role of BLIP-II in sporulation.     

New beta -lactamase inhibitory protein (BLIP-I) from Streptomyces exfoliatus SMF19 and its roles on the morphological differentiation

A new beta-lactamase inhibitory protein (BLIP-I) from Streptomyces exfoliatus SMF19 was purified and characterized. The molecular mass of BLIP-I was estimated to be 17.5 kDa by gel filtration fast protein liquid chromatography. The N-terminal sequence was NH(2)-Asn-Ser-Gly-Phe-Ser-Ala-Glu-Lys-Tyr-Glu-Gln-Ile-Gln-Phe-Gly. BLIP-I inhibited Bacto(R) Penase (Difco), and plasmid encoded TEM-1 beta-lactamase, whereas it did not inhibit Enterobacter cloacae beta-lactamases. The K(i) value of BLIP-I against TEM-1 beta-lactamase was determined to be 0.047 nm. The gene (bliA) encoding BLIP-I protein was identified by screening a genomic library using an oligonucleotide probe with a sequence based on the N-terminal sequence of BLIP-I. Analysis of the nucleotide sequence revealed that the gene was 558 base pairs in length and encoded a mature protein of 157 amino acid residues preceded by a 29-amino acid signal sequence. Pairwise comparison of the deduced amino acid sequence showed 38% identity with BLIP of Streptomyces clavuligerus. Furthermore, the 49th amino acid residue of BLIP-I was identical to Asp-49 of BLIP that was characterized to be an important residue for the inhibitory activity of BLIP. A modified BLIP-I in which Asp-49 was replaced by alanine (D49A) was obtained by site-directed mutagenesis. The inhibitory activities of recombinant (r) BLIP-I and its D49A mutant derivative, expressed in Escherichia coli, were compared. The K(i) value of rBLIP-I against TEM-1 beta-lactamase was similar to that of wild-type BLIP-I, but the D49A mutation increased the K(i) of rBLIP-I inhibition approximately 200-fold. A disruption mutant of the bliA gene in S. exfoliatus SMF19 was obtained by replacing the wild-type bliA gene with a copy inactivated by inserting a hygromycin resistance gene. The disruption mutant showed a bald phenotype, indicating that the bliA gene plays a role in morphological differentiation.     

Role of β-lactamase residues in a common interface for binding the structurally unrelated inhibitory proteins BLIP and BLIP-II

The β-lactamase inhibitory proteins (BLIPs) are a model system for examining molecular recognition in protein-protein interactions. BLIP and BLIP-II are structurally unrelated proteins that bind and inhibit TEM-1 β-lactamase. Both BLIPs share a common binding interface on TEM-1 and make contacts with many of the same TEM-1 surface residues. BLIP-II, however, binds TEM-1 over 150-fold tighter than BLIP despite the fact that it has fewer contact residues and a smaller binding interface. The role of eleven TEM-1 amino acid residues that contact both BLIP and BLIP-II was examined by alanine mutagenesis and determination of the association (k_on) and dissociation (k_off) rate constants for binding each partner. The substitutions had little impact on association rates and resulted in a wide range of dissociation rates as previously observed for substitutions on the BLIP side of the interface. The substitutions also had less effect on binding affinity for BLIP than BLIP-II. This is consistent with the high affinity and small binding interface of the TEM-1-BLIP-II complex, which predicts per residue contributions should be higher for TEM-1 binding to BLIP-II versus BLIP. Two TEM-1 residues (E104 and M129) were found to be hotspots for binding BLIP while five (L102, Y105, P107, K111, and M129) are hotspots for binding BLIP-II with only M129 as a common hotspot for both. Thus, although the same TEM-1 surface binds to both BLIP and BLIP-II, the distribution of binding energy on the surface is different for the two target proteins, that is, different binding strategies are employed.     

Calorimetric examination of high-affinity Src SH2 domain-tyrosyl phosphopeptide binding: dissection of the phosphopeptide sequence specificity and coupling energetics

SH2 domains are protein modules which interact with specific tyrosine phosphorylated sequences in target proteins. The SH2 domain of the Src kinase binds with high affinity to a tyrosine phosphorylated peptide containing the amino acids Glu, Glu, and Ile (EEI) at the positions +1, +2, and +3 C-terminal to the phosphotyrosine, respectively. To investigate the degree of selectivity of the Src SH2 domain for each amino acid of the EEI motif, the binding thermodynamics of a panel of substitutions at the +1 (Gln, Asp, Ala, Gly), +2 (Gln, Asp, Ala, Gly), and +3 (Leu, Val, Ala, Gly) positions were examined using titration microcalorimetry. It was revealed that the Src SH2 domain is insensitive (DeltaDeltaG degrees 相似文献   

An efficient heat-inducible Bacillus subtilis bacteriophage 105 expression and secretion system for the production of the Streptomyces clavuligerus beta-lactamase inhibitory protein (BLIP)

The Streptomyces clavuligerus beta-lactamase inhibitory protein (BLIP) has been shown to be a potent inhibitor of class A beta-lactamases including the Escherichia coli TEM-1 beta-lactamase (Ki = 0.6 nM). A heat-inducible BLIP expression system was constructed based on a derivative of Bacillus subtilis phage phi105. The recombinant BLIP produced by this system was secreted to the culture medium, purified to homogeneity, and fully active. We have shown that the signal peptide of BLIP functions well in B. subtilis to secrete BLIP out of the cells, which facilitates purification. The absence of a His-tag also avoids the activity and structure of BLIP being altered. An unprecedented high yield of recoverable protein in culture supernatant (3.6mg of >95% pure BLIP/l culture) was achieved by a simple purification protocol. We have developed an efficient production process in which the culture time before heat-induction was 3-4h and the culture supernatant could be collected 5h after induction. This total time of 8-9h is considered to be very short compared to that of the native S. clavuligerus culturing (60-70h). We achieved a very efficient BLIP production rate of 0.8-0.9mg/l/h. Heterologous gene expression was tightly controlled and no production of BLIP was observed before heat-induction, suggesting that cell density can be further increased to improve enzyme yield.     

Functional analyses of AmpC beta-lactamase through differential stability.

Despite decades of intense study, the complementarity of beta-lactams for beta-lactamases and penicillin binding proteins is poorly understood. For most of these enzymes, beta-lactam binding involves rapid formation of a covalent intermediate. This makes measuring the equilibrium between bound and free beta-lactam difficult, effectively precluding measurement of the interaction energy between the ligand and the enzyme. Here, we explore the energetic complementarity of beta-lactams for the beta-lactamase AmpC through reversible denaturation of adducts of the enzyme with beta-lactams. AmpC from Escherichia coli was reversibly denatured by temperature in a two-state manner with a temperature of melting (Tm) of 54.6 degrees C and a van't Hoff enthalpy of unfolding (deltaH(VH)) of 182 kcal/mol. Solvent denaturation gave a Gibbs free energy of unfolding in the absence of denaturant (deltaG(u)H2O) of 14.0 kcal/mol. Ligand binding perturbed the stability of the enzyme. The penicillin cloxacillin stabilized AmpC by 3.2 kcal/mol (deltaTm = +5.8 degrees C); the monobactam aztreonam stabilized the enzyme by 2.7 kcal/mol (deltaTm = +4.9 degrees C). Both acylating inhibitors complement the active site. Surprisingly, the oxacephem moxalactam and the carbapenem imipenem both destabilized AmpC, by 1.8 kcal/mol (deltaTm = -3.2 degrees C) and 0.7 kcal/mol (deltaTm = -1.2 degrees C), respectively. These beta-lactams, which share nonhydrogen substituents in the 6(7)alpha position of the beta-lactam ring, make unfavorable noncovalent interactions with the enzyme. Complexes of AmpC with transition state analog inhibitors were also reversibly denatured; both benzo(b)thiophene-2-boronic acid (BZBTH2B) and p-nitrophenyl phenylphosphonate (PNPP) stabilized AmpC. Finally, a catalytically inactive mutant of AmpC, Y150F, was reversibly denatured. It was 0.7 kcal/mol (deltaTm = -1.3 degrees C) less stable than wild-type (WT) by thermal denaturation. Both the cloxacillin and the moxalactam adducts with Y150F were significantly destabilized relative to their WT counterparts, suggesting that this residue plays a role in recognizing the acylated intermediate of the beta-lactamase reaction. Reversible denaturation allows for energetic analyses of the complementarity of AmpC for beta-lactams, through ligand binding, and for itself, through residue substitution. Reversible denaturation may be a useful way to study ligand complementarity to other beta-lactam binding proteins as well.     

Contribution of a single-turn alpha-helix to the conformational stability and activity of the alkaline proteinase inhibitor of Pseudomonas aeruginosa

The alkaline proteinase inhibitor of Pseudomonas aeruginosa (APRin), a high-affinity inhibitor of the serralysin family of bacterial metalloproteinases, is folded into an eight-stranded beta-barrel with an N-terminal trunk linked to the barrel by a single-turn alpha-helix (helix A, residues 8-11). We show here that deletion or modification of helix A decreases the conformational stability of APRin as assessed by thermal and chemical denaturation with guanidinium chloride (GdmCl). The apparent melting temperature T(m) of the wild-type protein was 81.5 degrees C at pH 7.1 as assessed by circular dichroism and 87.5 degrees C by differential scanning calorimetry. Reduction of the single disulfide bond of APRin decreased T(m) by approximately 18 degrees C, while deletion of residues 6-10 or 1-10 lowered T(m) by approximately 8 and approximately 14 degrees C, respectively. DeltaG(u) as assessed by chemical denaturation was 7.2 kcal mol(-)(1) at 25 degrees C for wild-type APRin and was decreased by 3.4, 2.4, and 2.6 kcal mol(-)(1) by disulfide reduction, deletion of residues 6-10, and deletion of residues 1-10, respectively. In contrast, deletion of residues 1-5 had no significant effect on either T(m) or DeltaG(u). Substitution of five helix-breaking Gly or Pro residues in positions 6-10 as well as disruption of hydrogen bonds involving residues within helix A (mutants Asp10Pro and Trp15Phe) also decreased T(m) and DeltaG(u). The data suggest that a hydrogen-bonding network involving Leu11 in helix A and Trp15 located at the top of the barrel may prevent access of solvent to the interior of the barrel. Disruption of the helix could facilitate solvation of the nonpolar interior of the barrel, thereby destabilizing its folded structure. Kinetic studies with single amino acid mutants in helix A indicate that it modulates the affinity of APRin for APR primarily by influencing the dissociation rate of the inhibitor from the complex.     

Noncovalent interaction energies in covalent complexes: TEM-1 beta-lactamase and beta-lactams

The class A beta-lactamase TEM-1 is a key bacterial resistance enzyme against beta-lactam antibiotics, but little is known about the energetic bases for complementarity between TEM-1 and its inhibitors. Most inhibitors form a covalent adduct with the catalytic Ser70, making the measurement of equilibrium constants, and hence interaction energies, technically difficult. This study evaluates noncovalent interactions within covalent complexes by examining the differential stability of TEM-1 and its inhibitor adducts. The thermal denaturation of TEM-1 follows a two-state, reversible model with a melting temperature (T(m)) of 51.6C and a van't Hoff enthalpy of unfolding (DeltaH(VH)) of 146.2 kcal/mol at pH 7.0. The stability of the enzyme changes on forming an inhibitor adduct. As expected, some inhibitors stabilize TEM-1; transition-state analogues increase the T(m) by up to 3.7C (1.7 kcal/mol). Surprisingly, all beta-lactam covalent acyl--enzyme complexes tested destabilize TEM-1 significantly relative to the apo-enzyme. For instance, the clinically used inhibitor clavulanic acid and the beta-lactamase-resistant beta-lactams moxalactam and imipenem destabilize TEM-1 by over 2.6C (1.2 kcal/mol) in their covalent adducts. Based on the structure of the TEM-1/imipenem complex (Maveyraud et al., J Am Chem Soc 1998;120:9748--52), destabilization by moxalactam and imipenem is thought to be caused by a steric clash between the side-chain of Asn132 and the 6(7)-alpha group of these beta-lactams. To test this hypothesis, the mutant enzyme N132A was made. In contrast with wild-type, the covalent complexes between N132A and both imipenem and moxalactam stabilize the enzyme, consistent with the hypothesis. To investigate the structural bases of this dramatic change in stability, the structure of N132A/imipenem was determined by X-ray crystallography. In the complex with N132A, imipenem adopts a very different conformation from that observed in the wild-type complex, and the putative destabilizing interaction with residue 132 is relieved. Studies of several enzymes suggest that beta-lactams, and covalent inhibitors in general, can have either net favorable or net unfavorable noncovalent interaction energies within the covalent complex. In the case of TEM-1, such unfavorable interactions convert substrate analogues into very effective inhibitors.     

Increasing protein stability by altering long-range coulombic interactions.

It is difficult to increase protein stability by adding hydrogen bonds or burying nonpolar surface. The results described here show that reversing the charge on a side chain on the surface of a protein is a useful way of increasing stability. Ribonuclease T1 is an acidic protein with a pI approximately 3.5 and a net charge of approximately -6 at pH 7. The side chain of Asp49 is hyperexposed, not hydrogen bonded, and 8 A from the nearest charged group. The stability of Asp49Ala is 0.5 kcal/mol greater than wild-type at pH 7 and 0.4 kcal/mol less at pH 2.5. The stability of Asp49His is 1.1 kcal/mol greater than wild-type at pH 6, where the histidine 49 side chain (pKa = 7.2) is positively charged. Similar results were obtained with ribonuclease Sa where Asp25Lys is 0.9 kcal/mol and Glu74Lys is 1.1 kcal/mol more stable than the wild-type enzyme. These results suggest that protein stability can be increased by improving the coulombic interactions among charged groups on the protein surface. In addition, the stability of RNase T1 decreases as more hydrophobic aromatic residues are substituted for Ala49, indicating a reverse hydrophobic effect.     

Detection of specific solvent rearrangement regions of an enzyme: NMR and ITC studies with aminoglycoside phosphotransferase(3')-IIIa

This work describes differential effects of solvent in complexes of the aminoglycoside phosphotransferase(3')-IIIa (APH) with different aminoglycosides and the detection of change in solvent structure at specific sites away from substrates. Binding of kanamycins to APH occurs with a larger negative DeltaH in H2O relative to D2O (DeltaDeltaH(H2O-D2O) < 0), while the reverse is true for neomycins. Unusually large negative DeltaCp values were observed for binding of aminoglycosides to APH. DeltaCp for the APH-neomycin complex was -1.6 kcal x mol(-1) x deg(-1). A break at 30 degrees C was observed in the APH-kanamycin complex yielding DeltaCp values of -0.7 kcal x mol(-1) x deg(-1) and -3.8 kcal x mol(-1) x deg(-1) below and above 30 degrees C, respectively. Neither the change in accessible surface area (DeltaASA) nor contributions from heats of ionization were sufficient to explain the large negative DeltaCp values. Most significantly, 15N-1H HSQC experiments showed that temperature-dependent shifts of the backbone amide protons of Leu 88, Ser 91, Cys 98, and Leu143 revealed a break at 30 degrees C only in the APH-kanamycin complex in spectra collected between 21 degrees C and 38 degrees C. These amino acids represent solvent reorganization sites that experience a change in solvent structure in their immediate environment as structurally different ligands bind to the enzyme. These residues were away from the substrate binding site and distributed in three hydrophobic patches in APH. Overall, our results show that a large number of factors affect DeltaCp and binding of structurally different ligand groups cause different solvent structure in the active site as well as differentially affecting specific sites away from the ligand binding site.     

State of binding subsites in Asp 101-modified lysozymes

The environments of the binding subsites in Asp 101-modified lysozyme, in which glucosamine or ethanolamine is covalently bound to the carboxyl group of Asp 101, were investigated by chemical modification and nuclear magnetic resonance spectroscopy. Trp 62 in each of the native and the modified lysozymes was nitrophenylsulfenylated. The yield of the nitrophenylsulfenylated derivative from the lysozyme modified with glucosamine at Asp 101 (GlcN-lysozyme) was considerably lower than those from native lysozyme and from the lysozyme modified with ethanolamine at Asp 101 (EtN-lysozyme). These results suggest that Trp 62 in GlcN-lysozyme is less susceptible to nitrophenylsulfenylation. Kinetic analyses of the [Trp 62 and Asp 101]-doubly modified lysozymes indicated that the nitrophenylsulfenylation of Trp 62 in the native lysozyme, EtN-lysozyme, or GlcN-lysozyme decreased the sugar residue affinity at subsite C while increasing the binding free energy change by 2.7 kcal/mol, 1.5 kcal/mol, or 0.1 kcal/mol, respectively. Although the profile of tryptophan indole NH resonances in the 1H-NMR spectrum for EtN-lysozyme was not different from that for the native lysozyme, the indole NH resonance of Trp 62 in GlcN-lysozyme was apparently perturbed in comparison with that of native lysozyme. These results suggest that the environment of subsite C in GlcN-lysozyme is considerably different from those in native lysozyme and EtN-lysozyme. The glucosamine residue attached to Asp 101 may contact the sugar residue binding site of the lysozyme, affecting the environment of subsite C.