Abstract
Cells adapt to different stress conditions, such as the antibiotics presence. This adaptation sometimes is achieved by changing relevant protein positions, of which the mutability is limited by structural constrains. Understanding the basis of these constrains represent an important challenge for both basic science and potential biotechnological applications. To study these constraints, we performed a systematic saturation mutagenesis of the transmembrane region of HokC, a toxin used by Escherichia coli to control its own population, and observed that 92% of single-point mutations are tolerated and that all the non-tolerated mutations have compensatory mutations that reverse their effect. We provide experimental evidence that HokC accumulates multiple compensatory mutations that are found as correlated mutations in the HokC family multiple sequence alignment. In agreement with these observations, transmembrane proteins show higher probability to present correlated mutations and are less densely packed locally than globular proteins; previous mutagenesis results on transmembrane proteins further support our observations on the high tolerability to mutations of transmembrane regions of proteins. Thus, our experimental results reveal the HokC transmembrane region high tolerance to loss-of-function mutations that is associated with low sequence conservation and high rate of correlated mutations in the HokC family sequences alignment, which are features shared with other transmembrane proteins.
Highlights
Understanding the structure–function relationship of proteins represents a challenge to design effective pharmacological compounds [1,2]
A common way to study the structure–function relationship of proteins involves the prediction of residues important for protein function based on the 3D structure of TM proteins, which are seldom available
In the absence of a 3D structure, critical residues for protein function may be predicted based on multiple sequence alignments (MSA) of similar proteins; MSA are built based on substitution matrices that, until recently, have been developed specific for TM proteins [5,6]
Summary
Understanding the structure–function relationship of proteins represents a challenge to design effective pharmacological compounds [1,2]. A common way to study the structure–function relationship of proteins involves the prediction of residues important for protein function based on the 3D structure of TM proteins, which are seldom available. We performed both experimental and computational analyses of a bitopic TM helical polypeptide, HokC This peptide is a toxin that kills Escherichia coli cells that express it [38], constituting a convenient system to identify critical residues for its toxic function (e.g., loss-of-function mutations will allow cells to growth). We provide experimental evidence that HokC accumulates multiple compensatory mutations that are found as correlated mutations in the HokC family multiple sequences alignment These correlated mutations are twice as much frequently found in transmembrane proteins than in the globular ones, which is accompanied by a lower local density of residue packing in transmembrane proteins compared with globular proteins. Our results together with previous experimental results support the idea that transmembrane proteins are more tolerant to loss-of-function mutations
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