Protein Variants Form a System of Networks: Microdiversity of IMP Metallo-Beta-Lactamases

Genome and metagenome sequencing projects support the view that only a tiny portion of the total protein microdiversity in the biosphere has been sequenced yet, while the vast majority of existing protein variants is still unknown. By using a network approach, the microdiversity of 42 metallo-β-lactamases of the IMP family was investigated. In the networks, the nodes are formed by the variants, while the edges correspond to single mutations between pairs of variants. The 42 variants were assigned to 7 separate networks. By analyzing the networks and their relationships, the structure of sequence space was studied and existing, but still unknown, functional variants were predicted. The largest network consists of 10 variants with IMP-1 in its center and includes two ubiquitous mutations, V67F and S262G. By relating the corresponding pairs of variants, the networks were integrated into a single system of networks. The largest network also included a quartet of variants: IMP-1, two single mutants, and the respective double mutant. The existence of quartets indicates that if two mutations resulted in functional enzymes, the double mutant may also be active and stable. Therefore, quartet construction from triplets was applied to predict 15 functional variants. Further functional mutants were predicted by applying the two ubiquitous mutations in all networks. In addition, since the networks are separated from each other by 10–15 mutations on average, it is expected that a subset of the theoretical intermediates are functional, and therefore are supposed to exist in the biosphere. Finally, the network analysis helps to distinguish between epistatic and additive effects of mutations; while the presence of correlated mutations indicates a strong interdependency between the respective positions, the mutations V67F and S262G are ubiquitous and therefore background independent.