Susceptibility of ß-lactamase to core amino acid substitutions

Joseph F. Petrosino1, Matthew Baker2 and Timothy Palzkill1,3,4

1 Department of Microbiology and Immunology, 2 Structural and Computational Biology and Molecular Biophysics Program and 3 Department of Biochemistry, Baylor College of Medicine, Houston, TX 77030, USA


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
To determine which amino acids in TEM-1 ß-lactamase are important for its structure and function, random libraries were previously constructed which systematically randomized the 263 codons of the mature enzyme. A comprehensive screening of these libraries identified several TEM-1 ß-lactamase core positions, including F66 and L76, which are strictly required for wild-type levels of hydrolytic activity. An examination of positions 66 and 76 in the class A ß-lactamase gene family shows that a phenylalanine at position 66 is strongly conserved while position 76 varies considerably among other ß-lactamases. It is possible that position 76 varies in the gene family because ß-lactamase mutants with non-conservative substitutions at position 76 retain partial function. In contrast, position 66 may remain unchanged in the gene family because non-conservative substitutions at this location are detrimental for enzyme structure and function. By determining the ß-lactam resistance levels of the 38 possible mutants at positions 66 and 76 in the TEM-1 enzyme, it was confirmed that position 76 is indeed more tolerant of non-conservative substitutions. An analysis of the Protein Data Bank files for three class A ß-lactamases indicates that volume constraints at position 66 are at least partly responsible for the low tolerance of substitutions at this position.

Keywords: ß-lactamase/compensating mutations/protein core/protein evolution/thermal value


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Amino acid interactions and packing in the protein core are critical for proper protein folding and function. While amino acid substitutions on the surface of a protein are generally tolerated, studies involving lambda repressor, T4 lysozyme and the globin family of proteins have identified several requirements for substitution of protein core amino acids (Lesk and Chothia, 1980Go; Bordo and Argos, 1989Go; Poteete et al., 1991Go; Rennell et al., 1991Go; Markiewicz et al., 1994Go; Terwilliger et al., 1994Go). These studies have shown that amino acid side-chain hydrophobicity, volume and steric constraints determine the ability of a substitution to be tolerated in the protein core. Of these parameters, hydrophobicity has been shown to be the most important core amino acid requisite with respect to maintaining protein structure and function (Lesk and Chothia, 1980Go; Bordo and Argos, 1989Go; Lim and Sauer, 1989Go). Conservative substitutions at buried positions, adhering to core hydrophobicity and volume constraints, will usually allow a protein to retain function. Stability and activity in these types of mutants are often maintained by repositioning the {alpha}-carbon backbone of neighboring amino acids, which enables the conservative mutation to be optimally repacked in the protein interior. Rearrangements in the protein core are therefore an effective means of accommodating mutations and may even result in a more tightly packed core than found in the original enzyme (Lesk and Chothia, 1980Go; Lim et al., 1994Go; Chothia and Gerstein, 1997Go).

A second mechanism by which core substitutions are tolerated is by the accumulation of compensatory mutations, which relieve the stress imparted by the original mutation (Poteete et al., 1991Go; Serrano et al., 1993Go; Baldwin et al., 1996Go). To date, examples of compensatory mutations in protein cores are relatively rare. This is presumably due to the need for multiple, specific substitutions to occur at positions surrounding the original mutation in order to suppress its defect (Lesk and Chothia, 1980Go; Baldwin et al., 1996Go). By the time the necessary compensatory mutations have arisen, it is likely that the original mutant would have been eliminated from the population. However, why certain core positions are still able to be compensated by secondary mutations remains unclear. A ß-lactamase model has been used to analyze how core substitutions are accommodated in a protein (Huang et al., 1996Go).

The most common mechanism of bacterial resistance to ß-lactam antibiotics, such as the penicillins and cephalosporins, is the production of ß-lactamases. These enzymes cleave the lactam ring of the antibiotic to render it inactive. Based on primary sequence homology, ß-lactamases have been grouped into four classes (Joris et al., 1988Go; Ambler et al., 1991Go). Classes A, C and D utilize a catalytic serine residue in the active site, while class B enzymes require a zinc atom for catalysis (Joris et al., 1988Go; Carfi et al., 1995Go). The crystal structures of several class A enzymes have been solved (Moews et al., 1990Go; Herzberg, 1991Go; Jelsch et al., 1993Go). The structures show that these enzymes share a similar three-dimensional structure and, along with primary sequence homology, suggest a common evolutionary origin for these proteins.

TEM-1 ß-lactamase, a class A enzyme, is the most prevalent plasmid encoded ß-lactamase found in Gram negative bacteria. Random replacement mutagenesis was previously used to completely randomize the 263 codons of the TEM-1 ß-lactamase gene (blaTEM-1) (Huang et al., 1996Go). This mutagenesis strategy targets three contiguous codons in blaTEM-1 and converts the wild-type sequence to all possible amino acid sequences within that three codon window (Huang et al., 1996Go). The set of all possible mutants for a given three amino acid window constitutes a random library. Eighty-eight random libraries encompassing the entire TEM-1 ß-lactamase gene were selected for mutants that confer wild-type levels of ampicillin resistance to Escherichia coli. Sequence data of functional mutants revealed that 43 of the 263 residues tested do not tolerate amino acid substitutions and are thought to be essential for TEM-1 ß-lactamase structure and function (Huang et al., 1996Go). Many of the conserved residues, such as F66 and L76, are core positions. This result is consistent with the general observation that core amino acids are less frequently substituted than surface residues (Bowie et al., 1990Go; Huang et al., 1996Go). However, while amino acid substitutions at residues F66 and L76 were conserved based on the in vitro mutagenesis results, the degree of sequence conservation at positions 66 and 76 in the class A ß-lactamase gene family is strikingly different (Figures 1 and 2GoGo). Residue 76 is substituted rather freely in other gene family members, while residue 66 is conserved in nearly all class A ß-lactamases. In the wild-type TEM-1 ß-lactamase structure, F66 and L76 are nearly equidistant from the active site pocket, lying approximately 12 and 10 Å, respectively, from the catalytic serine at position 70 (Figure 3Go) (from Brookhaven PDB entry 1BTL). F66 and L76 are both completely buried and interact with several surrounding amino acids (Jelsch et al., 1993Go). Therefore, upon initial inspection, the environments surrounding both F66 and L76 are similar. In other members of the class A ß-lactamase gene family, it appears that a series of mutations have evolved at surrounding positions to compensate for the non-conservative substitutions at position 76. As a result, hydrophobic interactions are maintained in enzymes having a hydrophobic amino acid at position 76, while a buried hydrogen bond network is found in enzymes having a hydrogen bond donor at position 76 (Huang et al., 1996Go).



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Fig. 1. Amino acids found at (A) positions 66 and (B) position 76 and at neighboring positions, in the TEM-1 selected random libraries, and in 20 class A ß-lactamase gene family members (Ambler et al., 1991Go; Huang et al., 1996Go). The TEM-1 ß-lactamase wild-type sequence is shown in boldface. Random library selected sequences are above the wild-type sequence, and class A ß-lactamase family member sequences are found below the wild-type sequence.

 


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Fig. 2. Conservation of positions 66 and 76 in the class A ß-lactamase gene family. Twenty-two class A ß-lactamases (or the strain a particular ß-lactamase originated from) are listed (Ambler et al., 1991Go).

 


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Fig. 3. Location of positions F66 and L76, with respect to S70, in TEM-1 ß-lactamase. Figure created with MOLSCRIPT (Kraulis, 1991Go).

 
There are two hypotheses that could explain why L76 has evolved through compensating mutations in the gene family and why F66 is relatively unchanged among class A ß-lactamases. The first hypothesis is that position 76 has evolved by chance and that position 66, also by chance, has not yet undergone significant variation. The second hypothesis is that even though no substitutions impart wild-type levels of TEM-1 ß-lactamase function, mutations at position 76 may retain significant levels of activity, while mutations at position 66 do not. If this is the case, then the probability that a compensatory mutation could occur before bacteria containing the original mutant were eliminated from the population would be much higher for a position 76 mutant. This hypothesis predicts that ß-lactamases with a substitution at position 76 retain significant function compared with ß-lactamases with a substitution at position 66. In this study, the second hypothesis was tested by examining the effect of mutations at positions 66 and 76. The activities of mutants with all 20 amino acids at both positions were determined. The antibiotic susceptibility and ß-lactamase hydrolytic activity levels of the 38 mutants and the wild-type TEM-1 enzyme confirmed that mutations at position 76 impart higher levels of activity than mutations at position 66.

In an attempt to determine why position 76 is more tolerant to substitutions than position 66, the thermal values for atoms surrounding and including both positions were examined in three class A ß-lactamases whose crystal structures have been solved: TEM-1 ß-lactamase, the PC1 ß-lactamase from Staphylococcus aureus and the ß-lactamase from Bacillus licheniformis 749/C (Moews et al., 1990Go; Herzberg, 1991Go; Jelsch et al., 1993Go). Both the PC1 and the 749/C ß-lactamases have hydrogen bond donors at position 76 (an asparagine and a threonine, respectively). Because thermal values reflect the range of movement a particular atom may have, they may explain why position 76 is more tolerant to substitution than position 66 (Petsko and Ringe, 1984Go). In a study involving 25 temperature-sensitive (ts) mutants of T4 lysozyme, it was found that all of the mutations altered amino acid side chains having lower than average thermal factors (Alber et al., 1987Go). It was concluded that amino acids with well-defined conformations form specific intramolecular interactions that make large contributions to the thermal stability of the protein (Alber et al., 1987Go). It was further interpreted that residues with high mobility or high solvent accessibility are much less susceptible to destabilizing substitutions, and that, in general, such amino acids make less of a contribution to protein stability (Alber et al., 1987Go). The actual number of atoms in contact with position 66 and 76 further shows how much structural constraint exists in these regions. It is presumed that the greater the number of contacts, the greater the constraint. The hypothesis is that there are relatively few atoms in contact with position 76 and these atoms have a wider range of motion than atoms surrounding position 66. As a result, position 76 would be more tolerant of substitutions because there is less constraint in this region. Meanwhile, it is expected that a greater number of atoms are in contact with position 66, and these atoms have lower thermal values. This would result in greater constraints on position 66 due to its environment. It was found that there is little correlation between the thermal values of atoms in contact with positions 66 and 76 and the ability of both positions to be substituted. Therefore, unlike the T4 lysozyme model, the thermal values and number of contacts around positions 66 and 76 do not explain why substitutions at position 76 are tolerated more than substitutions at position 66. However, additional analyses suggest a volume requirement may be at least partially responsible for the small tolerance of substitution at position 66.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Escherichia coli strains and plasmids

Escherichia coli BW313 [Hfr lysA(61–62), dut1, ung1, thi1, recA1, spoT1] was used to propagate plasmid DNA prior to mutagenesis (Kunkel et al., 1987Go). DNA was introduced into E.coli ES1301 [lacZ53, mutS201::Tn5, thyA36, rha5, metB1, deoC, IN (rrnD-rrnE)] immediately following mutagenesis (Siegel et al., 1982Go). Escherichia coli XL1-Blue [recA1, endA1, gyrA96, thi-1, hsdR17, supE44, relA1, lac, [F':: Tn10 (Tetr)proAB, {Delta}lacIq (lacZ)M15]] was used in determining antibiotic susceptibility and specific activity levels (Stratagene, Inc., Calif., USA). Mutagenesis was performed on the plasmid pBG66, which contains the wild-type blaTEM-1 gene and a cat gene encoding chloramphenicol acetyltransferase. This 4.8 kb plasmid also contains the f1 and ColEI origins of DNA replication (Palzkill and Botstein, 1992Go).

Random and site-directed mutagenesis

Oligonucleotide primers used for mutagenesis and DNA sequencing were synthesized by Genosys Biotechnologies, Inc., Texas, USA. The random library L76X was constructed (Huang and Palzkill, 1997Go). The F66X random library was constructed in the same manner using the following oligonucleotide: F66X, 5'-CTTTAAAAGTGCTCATCATTGGSNNACGTTCTTCGGGGCGAAAACTC-3', where N represents any base and S represents a C or a G. The mutagenesis template was pBG66 containing a SalI linker insertion at codon 65 of the blaTEM-1 gene (Huang et al., 1996Go). This SalI insert mutant also contained a single base deletion, rendering this mutant non-functional. Single-stranded DNA of the SalI insert mutant was used in the mutagenesis reaction, which was performed as previously described (Huang et al., 1996Go). Individual mutants from these libraries were obtained by streaking the libraries on LB agar plates containing 12.5 µg/ml chloramphenicol, and incubating the plates at 37°C for 18 h resulting in colonies containing the mutagenized pBG66 plasmid. Colonies from the chloramphenicol plates were replica plated onto duplicate 50, 100, 500 µg/ml and 1 mg/ml ampicillin LB agar plates. One set of plates was incubated 18 h at 37°C and the other at 30°C. This selection allowed various ß-lactamase-mediated phenotypes to be discerned by growth characteristics on the ampicillin plates at 30 and 37°C. The ß-lactamase genes from colonies growing under different ampicillin concentrations and temperatures were sequenced in attempt to isolate as many of the 19 different mutations possible for each residue. Fifteen of the 19 possible mutations at position 66 were isolated by sequencing 128 clones. Thirteen of the 19 possible mutations at position 76 were isolated by sequencing 48 clones. The F66D, F66E, F66G, F66P, L76A, L76D, L76E, L76F, L76K and L76Y mutants were constructed by oligonucleotide directed mutagenesis, as described by Kunkel et al. (1987). The sequence of these mutants was confirmed by the dideoxy chain termination method using PCR products as template (Hanke and Wink, 1994Go).

Antibiotic susceptibility

Minimum inhibitory concentrations (MICs) were determined by broth microdilution. Escherichia coli XL1-Blue cells harboring the pBG66 plasmid, either containing wild-type or a selected mutant ß-lactamase, were grown overnight in 2 ml 2xYT media supplemented with 12.5 µg/ml chloramphenicol. Each culture was diluted so that 1x104 cells could be inoculated into microtiter wells containing 100 µl LB media having twofold increases of each antibiotic tested (ampicillin and cephaloridine). After an 18 h incubation at 37°C, the plates were examined, and the lowest concentration of antibiotic that inhibited visual growth was recorded as the MIC.

Specific activity of ß-lactamase mutants

Escherichia coli XL1-Blue cells harboring the pBG66 plasmid, either containing wild-type or a selected mutant ß-lactamase, were grown overnight at 37°C in 2 ml of 2xYT medium supplemented with 12.5 µg/ml chloramphenicol. One hundred microliters of the overnight culture were used to inoculate 10 ml 2xYT with 12.5 µg/ml chloramphenicol. The 10 ml culture was grown to log phase (A600 = 0.5). The periplasmic contents of each culture were extracted using an osmotic shock procedure (Neu and Heppel, 1965Go). The total protein concentration of the periplasmic extract was determined by the method of Bradford (1976). ß-Lactamase activity assays were performed in 0.5 ml phosphate buffer (pH 7.0). Ampicillin (300 µM) or cephaloridine (1.0 M) was present in each reaction. Ampicillin hydrolysis was monitored as the loss of absorbance at 235 nm in a Beckman DU-640 spectrophotometer. Cephaloridine hydrolysis was monitored as the loss of absorbance at 260 nm. Specific activity was calculated as µM of antibiotic hydrolyzed per minute divided by the concentration of total protein in the reaction. The extinction coefficient used for ampicillin was {Delta}{varepsilon} = 900 M–1cm–1, and for cephaloridine was {Delta}{varepsilon} = 10 020 M–1cm–1 (Minami et al., 1980Go). All reactions were carried out at 30°C. Reaction mixtures were prewarmed at 30°C for 5 min before the addition of protein, and the reactions were monitored in the spectrophotometer using a water-jacketed, temperature controlled cuvette. Since all 40 mutants were not assayed at once, wild-type specific activity measurements were determined before each group of mutants to serve as an assay control. Because exact duplication of specific activity measurements is difficult, the percentage of wild-type activity for each given mutant was used for analysis.

Crystal contact and thermal value analysis

Potential hydrogen bond and van der Waals contact partners were identified by examining Brookhaven PDB data files 1BTL, 3BLM and 4BLM corresponding to TEM-1 ß-lactamase, the PC1 ß-lactamase from Staphylococcus aureus, and the ß-lactamase from Bacillus licheniformis 749/C, respectively (Moews et al., 1990Go; Herzberg, 1991Go; Jelsch et al., 1993Go). Atoms surrounding either position 66 or 76 were identified using the SYBYL software package (Tripos). Atoms within 4.0 Å of residue 66 and 76 were labeled as potential contact atoms, because they are within a reasonable van der Waals radius of the amino acid being examined. Atoms which may form hydrogen bonds with backbone nitrogen groups or the side chains of N76 in the PC-1 ß-lactamase and T76 in the 749/C enzyme were also included in the 4.0 Å radius.

The relative mobility of each atom in the X-ray crystal structures of TEM-1, PC1 and 749/C ß-lactamases is reflected by the B value of a particular atom. The B values for all of the atoms in contact with positions 66 and 76 in the ß-lactamase structures were obtained from the Brookhaven PDB files. The average thermal value for residues 66 and 76 in each model were determined by taking the sum of the thermal values for each atom in a particular amino acid and dividing by the number of atoms.

In order to compare packing density at F66 and L76, the three PDB files were modified so that they contained only the residues surrounding F66 and L76. The accessible surface in square Ångströms for all the residues within the structure subsets were then calculated (with respect to a probe sphere 1.40 Ångströms in diameter) using ACCESS (http://www.csb.yale.edu/download/download_descrip.html) (Lee and Richards, 1971Go). The volumes for these regions were then calculated from the output of ACCESS using VOLUME, under the default specifications (http://www.csb.yale.edu) (Richards, 1985Go). VOLUME determines the volume of a polyhedron surrounding selected atoms in a protein when the polyhedral faces are determined by one of three procedures based on the Voronoi construction (Richards, 1985Go).

To address the relative environment of F66 and L76 within the three structures, we analyzed the ability of positions 66 and 76 to accept other side chains using Rotamer Related, the option which counts and ranks position specific rotamers on the WHAT IF server (http://swift.embl-heidelberg.de/johnny/new/) (Vriend, 1990Go). The ranking system scores the most ideal amino acid a 1 and the amino acid furthest from ideal a 20.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Forty mutants representing all 20 amino acids at positions 66 and 76 were constructed as described in Materials and methods. Each was tested for its ability to confer resistance to ampicillin and cephaloridine. These substrates were chosen as representatives of two of the main classes of ß-lactam antibiotics, penicillins and cephalosporins. The length of time these antibiotics have been in use, and the fact that one is a penicillin and the other is a cephalosporin, suggest they are a fair representation of the antibiotic selective pressure that exists on bacteria carrying ß-lactamase.

According to the hypotheses described above, it was expected that a greater number of ß-lactamase mutants with substitutions at position 76 would confer significant levels of resistance to both antibiotics compared with mutants with substitutions at position 66. This proved to be the case as several position 76 ß-lactamase mutants did confer resistance to both ß-lactams (Table IGo). Since lower amounts of cephaloridine are toxic to TEM-1 ß-lactamase mutant-containing bacteria, the range of observed MICs is not as broad for the cephaloridine data as for the ampicillin data. Therefore, the MIC data for cephaloridine are not as discriminating as the data from the ampicillin MICs. The results from the cephaloridine MIC data, however, correlate well with the ampicillin data in that mutations which were most effective in conferring cephaloridine resistance also conferred the greatest resistance to ampicillin.


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Table I. MIC and enzyme specific activity measurements for position 66 and 76 TEM ß-lactamase mutants
 
As expected, hydrophobic substitutions at position 76 conferred the greatest amounts of resistance to both ß-lactams (Figure 4A and BGo). Meanwhile, position 66 tolerated very little change in hydrophobicity relative to the wild-type phenylalanine. In addition, non-hydrophobic substitutions at position 76 conferred significant levels of resistance to ampicillin and cephaloridine compared with non-hydrophobic substitutions at position 66. In particular, glutamate, proline or a histidine residue at position 76 all resulted in ß-lactamase enzymes that ranged from having partial to near wild-type activity. The tolerance of position 76 to polar amino acid substitutions is uncharacteristic of most core positions and indicates that position 76 is not a typical core position with respect to the variety of substitutions it tolerates (Bordo and Argos, 1989Go; Lim and Sauer, 1989Go).




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Fig. 4. Graphs of position 66 and position 76 mutant MICs for (A) ampicillin and (B) cephaloridine. Amino acids are listed in order of decreasing hydrophobicity with respect to the change in free energy from ethanol to water, with the most hydrophobic amino acids at the left end of the x-axis (Koolman and Rohm, 1996Go). Position 66 MICs are in open bars, and position 76 MICs are in shaded bars.

 
The specific activities of each of the 38 mutant enzymes, as well as the wild-type TEM-1 ß-lactamase, were determined to further support the antibiotic susceptibility data. As can be seen in Table IGo, the specific activity data corroborates the MIC data. The specific activities of several of the mutants that conferred low levels of antibiotic resistance were not determined because the amount of protein needed to measure activity exceeded the limits of the assay. In these cases, the inability to obtain specific activity measurements confirmed that these enzymes were as adversely affected by the indicated mutation as the MIC results suggested.

A direct comparison of the constraint existing in each environment was done by calculating the average thermal displacements for positions 66 and 76 in each of the ß-lactamases studied. The unidirectional mean square thermal displacement (µ2) is related to the crystallographic thermal factor B by the equation B = 8{pi}22). The B values are comprised of contributions made by thermal motion, segmental motion, lattice disorder and errors in the molecular model. However, thermal motion has been found to dominate the observed B values in several protein X-ray crystal structures (Petsko and Ringe, 1984Go). The ability of position 76 to tolerate more substitutions than position 66 in the protein core leads to the prediction that there are fewer structural constraints around position 76. The sum of the thermal factors for atoms from each amino acid, at both positions, was divided by the number of atoms in the residue, and the resulting averages were compared. It was discovered that the average thermal factor of position 66 was slightly higher than the average thermal factor at position 76 in all three ß-lactamases (Table IIGo). Therefore, according to the average thermal factor parameter, there does not appear to be a significant difference in the constraint found at positions 66 and 76.


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Table II. Average B values (main chain and side chain atoms), and standard deviations of amino acids occupying positions 66 and 76 in the TEM-1, PC1 and 749/C ß-lactamases
 
To investigate further why positions 66 and 76 have different tolerances to substitutions, the side-chain contacts and thermal values for each of three ß-lactamases, for which X-ray structures are available, were determined as described in the Materials and methods. By examining the atoms in contact with positions 66 and 76, slight differences in the environments surrounding each residue may be found. It is not possible, from the crystal structures, to determine which of the contact atom(s) makes the greatest contribution to the positioning of any one amino acid. Therefore, any atoms within possible van der Waals contact (4 Å) of residue 66 or 76 were included in the data sets. In the TEM-1, PC1 and 749/C ß-lactamases, surrounding residues appear to make a similar number of contacts at position 66 compared with position 76 (Table IIIGo). In TEM-1, both F66 and L76 are within van der Waals contact of 26 surrounding atoms (Figure 5A and BGo). In the PC1 enzyme, F66 is within van der Waals contact of 33 surrounding atoms, and N76 is within contact distance of 29 atoms (Figure 6A and BGo). In the 749/C enzyme, F66 is within van der Waals contact of 32 atoms, while T76 is within contact distance of 30 atoms (Figure 7A and BGo). Based on the number of contact atoms, the amount of constraint at positions 66 and 76 appears nearly equivalent in all three ß-lactamases. A slightly greater amount of constraint at position 66 may exist in the PC1 and 749/C enzymes; however, there is not a dramatic difference in the number of potential contact atoms between positions 66 and 76 in these ß-lactamases. Furthermore, there appears to be no strong correlation between positions 66 and 76 with regards to the number of contacts made to main chain atoms versus side chain atoms. Overall, it appears that the greater tolerance of substitutions at position 76, compared with position 66, is not dependent on the number of atoms in contact with adjacent amino acids at both positions.


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Table III. Amino acids in contact with positions 66 and 76 in the TEM-1, PC1 and 749/C ß-lactamases.
 


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Fig. 5. Diagrams of the position 66 and 76 regions in TEM-1 ß-lactamase. (A) Residues within 4 Å of F66. (B) Residues within 4 Å of L76.

 


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Fig. 6. Diagrams of the position 66 and 76 regions in PC1 ß-lactamase. (A) Residues within 4 Å of F66. (B) Residues within 4 Å of N76.

 


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Fig. 7. Diagrams of the position 66 and 76 regions in the ß-lactamase from Bacillus licheniformis 749/C. (A) Residues within 4 Å of F66. (B) Residues within 4 Å of T76.

 
Even though the number of atoms in contact with positions 66 and 76 in the three ß-lactamases are nearly equal, it is still possible that the atoms in contact with position 66 are under greater constraint compared with the atoms in contact with position 76. To test this theory, the B values of atoms in contact with positions 66 and 76 were used to compare relative levels of constraint imparted by the atoms in contact with positions 66 and 76 in the three ß-lactamases examined. It was expected that the B values of contact atoms surrounding position 76 would be greater than the B values of contact atoms surrounding position 66 in each ß-lactamase. However, like the number of contact atoms at each position, the average B values of atoms in contact with positions 66 and 76 are nearly equal in each of the three ß-lactamases examined (Table IIIGo). Based on the average B values and their standard deviations, there appears to be little difference in the thermal displacement between positions 66 and 76. Therefore, there is no correlation between the ability of positions 66 and 76 to be substituted in the three enzymes and the thermal values of atoms in contact with each of these positions.

Structural constraint was further examined by comparing the position 66 and 76 packing densities, as measured by the Voronoi volumes at, and surrounding, both positions. If a correlation between the Voronoi volumes at positions 66 and 76 exists, it would suggest that there is a volume requirement for the amino acids that may occupy these positions. No correlation was observed regarding Voronoi volumes of the environments surrounding both positions; however, a strong correlation between the volumes of the amino acids occupying positions 66 and 76 was found (Table IVGo). In each of the three ß-lactamases, position 66 occupies a greater volume than position 76.


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Table IV. Voronoi volumes of position 66 and 76, and the environments around these positions in the three ß-lactamases examined
 
To further characterize the types of amino acids preferred at positions 66 and 76, the rotamer distribution was counted for positions 66 and 76 in the three ß-lactamases tested. The output of the Rotamer Related command from the WHAT IF server ranks the ability of an amino acid to be incorporated at a given position in a polypeptide (Vriend, 1990Go). The data generated suggests that the phenylalanine and leucine at positions 66 and 76 in the TEM-1 enzyme are highly optimized since both are predicted to be the second best amino acids able to occupy those locations (Table VGo). Meanwhile, the residues at both positions in the other two enzymes are not as ideal according to the predicted ranking. While N76 is not as favored as F66 in the PC1 enzyme (14th versus 7th ranked), T76 and F66 are almost equally ranked among preferred residues at those positions in the 749/C enzyme (8th versus 7th).


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Table V. Ranking of residues preferred at positions 66 and 76 in the three ß-lactamases tested as determined by rotamer repacking
 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
From in vitro mutagenesis experiments, it was previously found that substitutions at core residue 76 were unable to confer wild-type hydrolytic activity in TEM-1 ß-lactamase, yet in class A ß-lactamases non-conservative substitutions are found at position 76 among gene family members (Ambler et al., 1991Go; Huang et al., 1996Go). Positions adjacent to residue 76 in the three-dimensional structure of the enzyme appear to compensate for non-conservative substitutions in the gene family. For example, in the S.aureus and B.licheniformis ß-lactamases, an asparagine and a threonine are located at position 76, respectively. The amino acids and contact atoms surrounding these residues are hydrophilic in both enzymes, suggesting that a buried hydrogen bond network has replaced the hydrophobic interactions observed in the TEM-1 enzyme. Since the class A enzymes are believed to have evolved from a single precursor, it is thought that this change in the local environment is due to coupled, compensating mutations in the position 76 region (Huang et al., 1996Go). Therefore, position 76 is an excellent example of localized evolution within a protein core. In contrast, F66 is predominantly conserved throughout the class A gene family and also could not tolerate amino acid substitutions in mutagenesis studies (Huang et al., 1996Go). Two class A ß-lactamases have been found that contain a tyrosine at position 66 rather than a phenylalanine, the CfxA enzyme from Bacteriodes vulgatus and the CepA enzyme of Bacteriodes fragilis. A comparison of the amino acid sequences of these two enzymes suggests that these ß-lactamases may have diverged from the class A gene family early in their evolution (Parker and Smith, 1993Go; Rogers et al., 1993Go). It is likely that the differences between the CfxA and CepA enzymes and the class A ß-lactamases are responsible for the tolerance of a tyrosine at position 66.

To test the relative tolerance of positions 66 and 76 to substitution in the TEM-1 enzyme, we constructed mutants containing every amino acid substitution at both sites and examined the ability of these mutants to confer bacterial resistance to two common ß-lactam antibiotics. The antibiotic susceptibility studies showed that mutations at position 76 do appear to be less deleterious than mutations at position 66 in TEM-1 ß-lactamase. Typical of core residues, position 76, and to a lesser extent, position 66 were tolerant of mutations in which the replacement amino acids were hydrophobic and were within one methyl group in volume change. However, several non-conservative hydrophilic amino acid substitutions and other small volume amino acid substitutions at position 76 resulted in mutant ß-lactamases with significant levels of activity. Of these non-conservative substitutions conferring partial activity, glutamate is the only one found at position 76 in a class A enzyme, the blaU gene from Streptomyces cacaoi. It was concluded that the ability of position 76 to tolerate several non-conservative substitutions and still retain partial activity is directly correlated to the ability of compensating mutations to arise in response to position 76 mutations. Many of the possible mutations at position 76 will not completely eliminate enzyme activity, and the chance of secondary mutations arising to restore the mutant to wild-type levels of activity is increased.

If a non-conservative mutation is to be maintained in the protein core, subsequent compensatory mutations are required. Because protein cores are believed to evolve through a constrained, step-by-step process that does not dramatically affect the main-chain fold, the probability that a compensating mutation will arise before the original mutant is eliminated from a population is low. However, if the original substitution does not significantly compromise the protein's structure or function, then the probability that a secondary mutation will arise to compensate for the original substitution before it is eliminated is increased (Bordo and Argos, 1989Go; Lim and Sauer, 1989Go; Malcolm et al., 1990Go; Baldwin et al., 1996Go). An analysis of T4 lysozyme has shown that even when interacting buried residues are exchanged, complete compensation is not achieved (Baldwin et al., 1996Go). Therefore, it appears that several mutations are needed to reconstitute the activity of the wild-type protein. One conclusion from the lysozyme study was that a reversion of a deleterious mutation back to the wild-type residue would be the easiest way for a protein to compensate for a non-conservative substitution rather than a series of secondary mutations. The results described here also suggest that a reversion mutation would be the easiest and possibly the only way to compensate for a non-conservative substitution at a typical core position, such as F66. However, if the core position is able to retain partial activity in response to a non-conservative substitution, then second-site compensating mutations would provide a feasible pathway to restore wild-type activity levels as well.

In order to determine why substitutions at position 76 are less detrimental to ß-lactamase structure, the environments around positions 66 and 76 were analyzed. By calculating the average thermal factors of positions 66 and 76 in the three ß-lactamases examined, it appears that the amount of structural constraint at both positions in each enzyme are similar. Furthermore, in the three ß-lactamases tested, the amino acid occupying position 76 makes a similar number of van der Waals or hydrogen bond contacts compared with F66. Those residues making contact with position 76 also have similar thermal values compared with the F66 contact residues. Both of these results suggest that there are equivalent structural constraints on positions 66 and 76. However, the Voronoi volumes occupied by positions 66 and 76 in the three enzymes does suggest that there is a requirement for larger amino acids at position 66. This agrees favorably with our mutant activity data which shows that larger amino acids are able to confer partial activity to position 66 mutants, while a variety of amino acids may confer partial activity to position 76 mutants. The rotamer re-packing data provides an estimate of how optimal the existing amino acids are at each of the positions tested. It appears that the core of the TEM-1 enzyme is the most evolved of the three enzymes examined because both F66 and L76 rank second in preferred amino acids at these positions. Meanwhile, the wild-type amino acids at positions 66 and 76 in the PC1 and 749/C enzymes rank no higher than 7th. Particularly interesting is the fact that the asparagine at position 76 in the PC1 enzyme is ranked 14th suggesting that substitutions for this residue would be tolerated, and in many cases, preferred. However, these results may also reflect a requirement for asparagine at position 76 for a folding pathway or other purpose.

The extent that hydrophobicity, side chain volume and steric hindrance contribute to protein core constraint has been well documented (Lesk and Chothia, 1980Go; Bordo and Argos, 1989Go; Poteete et al., 1991Go; Rennell et al., 1991Go; Markiewicz et al., 1994Go; Terwilliger et al., 1994Go). Additional studies of the Cro protein of phage {lambda}, staphylococcal nuclease and kanamycin nucleotidyltransferase have shown that there is no simple pattern to which these properties contribute to protein stability (Shortle and Lin, 1985Go; Matsumura et al., 1986Go; Pakula et al., 1986Go). A correlation between low thermal factors, fractional accessible surface area and detrimental amino acid substitutions has demonstrated the importance of these two parameters as well (Alber et al., 1987Go). In the case of positions 66 and 76 in the three ß-lactamases examined, side chain volume requirements may explain why position 66 tolerates fewer substitutions than position 76.

It is not clear whether position 76 was the first amino acid to be substituted in the PC1 or 749/C ß-lactamases, or if a neighboring position mutated first and a secondary mutation occurred at position 76 to accommodate the adjacent hydrogen bonding amino acid. Positions 66 and 76 are both surrounded by amino acids that display various degrees of conservation in the class A ß-lactamase gene family as well as in the random library selection reported previously (Huang et al., 1996Go). Even though the environments surrounding positions 66 and 76 appear similar, there are explanations which could further account for the conservation of F66 in the class A ß-lactamase gene family. For example, F66 may play an important role in the folding of ß-lactamase while position 76 does not. It is also possible that among the atoms within contact distance of position 66, there are interactions which cannot be disrupted. Such important contact(s) may not exist at position 76.


    Acknowledgments
 
We would like to thank Dr Wanzhi Huang for the construction of the L76X library. This work was supported by the National Institutes of Health Grant AI32956.


    Notes
 
4 To whom correspondence should be addressed Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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Received February 1, 1999; revised May 18, 1999; accepted June 4, 1999.





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