Mutational scanning reveals the determinants of protein insertion and association energetics in the plasma membrane.

Insertion of helix-forming segments into the membrane and their association determines the structure, function, and expression levels of all plasma membrane proteins. However, systematic and reliable quantification of membrane-protein energetics has been challenging. We developed a deep mutational scanning method to monitor the effects of hundreds of point mutations on helix insertion and self-association within the bacterial inner membrane. The assay quantifies insertion energetics for all natural amino acids at 27 positions across the membrane, revealing that the hydrophobicity of biological membranes is significantly higher than appreciated. We further quantitate the contributions to membrane-protein insertion from positively charged residues at the cytoplasm-membrane interface and reveal large and unanticipated differences among these residues. Finally, we derive comprehensive mutational landscapes in the membrane domains of Glycophorin A and the ErbB2 oncogene, and find that insertion and self-association are strongly coupled in receptor homodimers.

Computational protein design suggests that human PCNA-partner interactions are not optimized for affinity.

Increasing the affinity of binding proteins is invaluable for basic and applied biological research. Currently, directed protein evolution experiments are the main approach for generating such proteins through the construction and screening of large mutant libraries. Proliferating cell nuclear antigen (PCNA) is an essential hub protein that interacts with many different partners to tightly regulate DNA replication and repair in all eukaryotes. Here, we used computational design to generate human PCNA mutants with enhanced affinity for several different partners. We identified double mutations in PCNA, outside the main partner binding site, that were predicted to increase PCNA-partner binding affinities compared to the wild-type protein by forming additional hydrophobic interactions with conserved residues in the PCNA partners. Affinity increases were experimentally validated with four different PCNA partners, demonstrating that computational design can reveal unexpected regions where affinity enhancements in natural systems are possible. The designed PCNA mutants can be used as a valuable tool for further examination of the regulation of PCNA-partner interactions during DNA replication and repair both in vitro and in vivo. More broadly, the ability to engineer affinity increases toward several PCNA partners suggests that interaction affinity is not an evolutionarily optimized trait of this system.

Tight coevolution of proliferating cell nuclear antigen (PCNA)-partner interaction networks in fungi leads to interspecies network incompatibility.

The structure and connectivity of protein-protein interaction (PPI) networks are maintained throughout evolution by coordinated changes (coevolution) of network proteins. Despite extensive research, relatively little is known regarding the molecular basis and functional implications of the coevolution of PPI networks. Here, we used proliferating cell nuclear antigen, a hub protein that mediates DNA replication and repair in eukaryotes, as a model system to study the coevolution of PPI networks in fungi. Using a combined bioinformatics and experimental approach, we discovered that PCNA-partner interactions tightly coevolved in fungal species, leading to specific modes of recognition. We found that fungal proliferating cell nuclear antigen-partner interaction networks diverged into two distinct groups as a result of such coevolution and that hybrid networks of these groups are functionally noncompatible in Saccharomyces cerevisiae. Our results indicate that the coevolution of PPI networks can form functional barriers between fungal species, and thus can promote and fix speciation.

Subtle alterations in PCNA-partner interactions severely impair DNA replication and repair.

The robustness of complex biological processes in the face of environmental and genetic perturbations is a key biological trait. However, while robustness has been extensively studied, little is known regarding the fragility of biological processes. Here, we have examined the susceptibility of DNA replication and repair processes mediated by the proliferating cell nuclear antigen (PCNA). Using protein directed evolution, biochemical, and genetic approaches, we have generated and characterized PCNA mutants with increased affinity for several key partners of the PCNA-partner network. We found that increases in PCNA-partner interaction affinities led to severe in vivo phenotypic defects. Surprisingly, such defects are much more severe than those induced by complete abolishment of the respective interactions. Thus, the subtle and tunable nature of these affinity perturbations produced different phenotypic effects than realized with traditional "on-off" analysis using gene knockouts. Our findings indicate that biological systems can be robust to one set of perturbations yet fragile to others.

Coevolution predicts direct interactions between mtDNA-encoded and nDNA-encoded subunits of oxidative phosphorylation complex i.

Despite years of research, the structure of the largest mammalian oxidative phosphorylation (OXPHOS) complex, NADH-ubiquinone oxidoreductase (complex I), and the interactions among its 45 subunits are not fully understood. Since complex I harbors subunits encoded by mitochondrial DNA (mtDNA) and nuclear DNA (nDNA) genomes, with the former evolving ∼10 times faster than the latter, tight cytonuclear coevolution is expected and observed. Recently, we identified three nDNA-encoded complex I subunits that underwent accelerated amino acid replacement, suggesting their adjustment to the elevated mtDNA rate of change. Hence, they constitute excellent candidates for binding mtDNA-encoded subunits. Here, we further disentangle the network of physical cytonuclear interactions within complex I by analyzing subunits coevolution. Firstly, relying on the bioinformatic analysis of 10 protein complexes possessing solved structures, we show that signals of coevolution identified physically interacting subunits with nearly 90% accuracy, thus lending support to our approach. When applying this approach to cytonuclear interaction within complex I, we predict that the 'rate-accelerated' nDNA-encoded subunits of complex I, NDUFC2 and NDUFA1, likely interact with the mtDNA-encoded subunits ND5/ND4 and ND5/ND4/ND1, respectively. Furthermore, we predicted interactions among mtDNA-encoded complex I subunits. Using the yeast two-hybrid system, we experimentally confirmed the predicted interactions of human NDUFC2 with ND4, the interactions of human NDUFA1 with ND1 and ND4, and the lack of interaction of NDUFC2 with ND3 and NDUFA1, thus providing a proof of concept for our approach. Our study shows, for the first time, evidence for direct interactions between nDNA-encoded and mtDNA-encoded subunits of human OXPHOS complex I and paves the path towards deciphering subunit interactions within complexes lacking three-dimensional structures. Our subunit-interactions-predicting method, ComplexCorr, is available at http://webclu.bio.wzw.tum.de/complexcorr.

Surface display of redox enzymes in microbial fuel cells.

A novel concept for a biofuel cell is presented. Enzyme based fuel cells suffer from enzyme instability when a long time of operation is required. Hence, a system that will continuously produce the biocatalyst needed for the system is necessary. A hybrid of an enzyme-based microbial fuel cell was developed. The redox enzyme glucose oxidase from Aspergillus niger was displayed on the surface of Saccharomyces cerevisiae using the Yeast Surface Display System in a high copy number and as an active enzyme. We have demonstrated its activity both biochemically and electrochemically and observed much higher activity over yeast cells not displaying glucose oxidase as well as over purified glucose oxidase from Aspergillus niger. Further, we were able to construct a biofuel cell, where the anode was comprised of the yeast cells displaying glucose oxidase in the presence of a mediator (methylene blue) and the cathode compartment was comprised of the oxygen reducing enzyme laccase from Trametes versicolor and a redox mediator. Our constructed biofuel cell displayed higher power outputs and current densities than those observed for unmodified yeast and a much longer time of operation in comparison with a similar cell where the anode is comprised of purified glucose oxidase.