Fitness Effects of Single Amino Acid Insertions and Deletions in TEM-1 β-Lactamase

Abstract

Short insertions and deletions (InDels) are a common type of mutation found in nature and a useful source of variation in protein engineering. InDel events have important consequences in protein evolution, often opening new pathways for adaptation. Yet much less is known about the effects of InDels compared to point mutations and amino acid substitutions. In particular, deep mutagenesis studies on the distribution of fitness effects of mutations have focused almost exclusively on amino acid substitutions. Here, we present a near-comprehensive analysis of the fitness effects of single amino acid InDels in TEM-1 β-lactamase. While we found InDels to be largely deleterious, partially overlapping deletion-tolerant and insertion-tolerant regions were observed throughout the protein, especially in unstructured regions and at the end of helices. The signal sequence of TEM-1 tolerated InDels more than the mature protein. Most regions of the protein tolerated insertions more than deletions, but a few regions tolerated deletions more than insertions. We examined the relationship between InDel tolerance and a variety of measures to help understand its origin. These measures included evolutionary variation in β-lactamases, secondary structure identity, tolerance to amino acid substitutions, solvent accessibility, and sidechain weighted contact number. We found secondary structure, weighted contact number, and evolutionary variation in class A beta-lactamases to be the somewhat predictive of InDel fitness effects.

Accessing unexplored regions of sequence space in directed enzyme evolution via insertion/deletion mutagenesis

“…Indeed, this view is now experimentally corroborated by our work on wt PTE (Fig. 6 ) as well as a recent deep mutational scanning study investigating the fitness effects of single amino acid InDels on TEM-1 β-lactamase 44 . However, InDels have also been shown to be contribute to functional divergence in several enzyme families, such as lactate and malate dehydrogenases 45 , tRNA nucleotidyltransferases 46 , nitroreductases 47 , o-succinylbenzoate synthases 43 and phosphotriesterase-like lactonases 4 , 48 .…”

Access to unexplored regions of sequence space in directed enzyme evolutionviainsertion/deletion mutagenesis

“…A corollary of the comparatively drastic effect of InDels on protein structure is the perception that they are more deleterious. Indeed, this view is now experimentally corroborated by our work on wtPTE ( Figure 5) as well as a recent deep mutational scanning study investigating the fitness effects of single amino acid InDels on TEM-1 β-lactamase (54). However, InDels have also been shown to be contribute to functional divergence in several enzyme families, such as lactate and malate dehydrogenases (55), tRNA nucleotidyltransferases (56), nitroreductases (57), osuccinylbenzoate synthases (53), and phosphotriesterase-like lactonases (2,4).…”

KPC Beta-Lactamases Are Permissive to Insertions and Deletions Conferring Substrate Spectrum Modifications and Resistance to Ceftazidime-Avibactam

“…The 3 main loops surrounding the active site are affected either by insertions or deletions, reinforcing the idea that loops are more tolerant to indels, especially the omega-loop. Mutations in the omega-loop can modify salt bridges (specifically between Arg 164 and Asp 179 ) and, consequently, enhance the flexibility of the loop, widening the substrate spectrum (23,24).…”