Deep sequencing of systematic combinatorial libraries reveals β-lactamase sequence constraints at high resolution

Abstract

In this study, combinatorial libraries were used in conjunction with ultrahigh-throughput sequencing to comprehensively determine the impact of each of the 19 possible amino acid substitutions at each residue position in the TEM-1 β-lactamase enzyme. The libraries were introduced into Escherichiacoli, and mutants were selected for ampicillin resistance. The selected colonies were pooled and subjected to ultrahigh-throughput sequencing to reveal the sequence preferences at each position. The depth of sequencing provided a clear, statistically significant picture of what amino acids are favored for ampicillin hydrolysis for all 263 positions of the enzyme in one experiment. Although the enzyme is generally tolerant of amino acid substitutions, several surface positions far from the active site are sensitive to substitutions suggesting a role for these residues in enzyme stability, solubility, or catalysis. In addition, information on the frequency of substitutions was used to identify mutations that increase enzyme thermodynamic stability. Finally, a comparison of sequence requirements based on the mutagenesis results versus those inferred from sequence conservation in an alignment of 156 class A β-lactamases reveals significant differences in that several residues in TEM-1 do not tolerate substitutions and yet extensive variation is observed in the alignment and vice versa. An analysis of the TEM-1 and other class A structures suggests that residues that vary in the alignment may nevertheless make unique, but important, interactions within individual enzymes.

Figure 1

Position of random libraries on TEM-1 β-lactamase sequence. The nucleotides randomized for each library are boxed. The amino acids randomized for each library are indicated above each box.

Figure 2

Ultra-high throughput sequencing of TEM-1 β-lactamase random libraries. A. 158-HVT-160 random library and list of sequences of ampicillin resistant clones obtained by 454 DNA sequencing. A total of 8635 sequence reads were obtained. B. Summary of 158-160 library sequencing results. The wild type sequence is at top and the amino acids found among ampicillin resistant clones are listed below. The number of times an amino acid type occurred is indicated by the superscript number. C. 242-GSR-244 library and list of ampicillin resistant clone sequences. D. Summary of 242-244 library sequencing results. E. Location of amino acid residues 158-160 and 242-244 on the TEM-1 β-lactamase structure (PDB code: 1BTL). A ribbon diagram of the TEM-1 structure is shown with the highlighted boxes containing a side chain view of the regions. The active site residue Ser70 is indicated in yellow.

Figure 3

Graph indicating the effective number of amino acid substitutions (k*) at each residue position in TEM-1 β-lactamase that are consistent with high levels of ampicillin resistance. Positions with low k* values do not tolerate substitutions while positions with high k* values can accept many different substitutions and retain high levels of function.

Figure 4

Summary of the effective number of amino acid substitutions (k*) on the structure of TEM-1 β-lactamase. At left is a view of enzyme with the active site serine 70 indicated with an arrow. At right is a 180^o rotation of the structure. The color of the amino acid residues is based on the observed k* values. Red, k*<5; yellow, k*<10; green, k*>10.

Figure 5

Determination of ampicillin resistance levels of wild type and substitutions of surface charged residues in TEM-1 β-lactamase. The ampicillin resistance level of E. coli containing each mutant was measured by spotting serial dilutions of cultures containing each mutant on agar plates containing increasing concentrations of ampicillin. The maximum dilution for which growth of colonies occurred is indicated on the y-axis versus the concentration of ampicillin in the agar plates on the x-axis.

Figure 6

Heat map representation of ΔΔG^stat values for each randomized position in TEM-1 β^-lactamase. The X-axis lists each residue position while the Y-axis indicates each of the two amino acid types. Each column above the residue position is therefore the set of ΔΔG^stat values for each amino acid substitution. The correspondence between heat map color and ΔΔG^stat is shown at lower right. Positive ΔΔG^stat values indicate a residue occurs at a frequency lower than the frequency of the wild type residue (red) while a negative value indicates the residue occurs more frequently than the wild type (green) residue among the sequenced ampicillin resistant clones. The order of the amino acid rows (Y-axis) were clustered by comparison of the ΔΔG^stat patterns for each amino acid type. Residues that are in the same branches of the tree exhibit similar effects on ΔΔG^stat values.Pearson correlation coefficients are shown adjacent to the hierarchical tree of amino acid substitutions.

Figure 7

Thermal denaturation curves of wild type TEM-1 and selected β-lactamase variants obtained from circular dichroism measurements at increasing temperatures. Fractional changes in the CD signal are shown for the wild-type (black), V31R (red), and G78A (green).

Figure 8

Comparison of the effective number of substitutions (k*) determined from TEM-1 β-lactamase mutagenesis experiments versus an alignment of class A β-lactamase sequences. A. Bar graph indicated the difference in k* between the mutagenesis experiments and the class A enzyme alignment. The values shown are k* mutagenesis - k* alignment. B. Plot of k* values determined based on the class A alignment versus the random mutagenesis results. Analysis reveals a correlation coefficient r² of 0.22 with a P value <0.0001.

Figure 9

Comparison of TEM-1 and CTX-M-16 β-lactamase structures at positions 32 and 290 showing the difference in residue environment in the different class A enzymes. A. Molecular environment of TEM-1 β-lactamase residues Trp290 and Arg259 (PBP ID 1BTL). B. Molecular environment of residue 290 in CTX-M-16 β-lactamase (PDB ID 1YLW). C. Environment of TEM-1 β-lactamase residue Lys32. D. Environment of CTX-M-16 β-lactamase residue 32.