Search

The majority of protein interactions in most organisms are unknown, and experimental methods for determining protein interactions can yield divergent results. Here we use an orthogonal, purely computational method based on sequence coevolution to discover protein interactions at large scale. In the model organism Escherichia coli, 53% of protein pairs in the proteome are eligible for our method given currently available sequenced genomes. When assaying the entire cell envelope proteome, which is understudied due to experimental challenges, we found 620 likely interactions and their predicted structures, increasing the space of known interactions by 529. Our results show that genomic sequencing data can be used to predict and resolve protein interactions to atomic resolution at large scale. Predictions and code are freely available at https://marks.hms.harvard.edu/ecolicomplex and https://github.com/debbiemarkslab/EVcouplings

Clustered protocadherins, a large family of paralogous proteins that play important roles in neuronal development, provide an important case study of interaction specificity in a large eukaryotic protein family. A mammalian genome has more than 50 clustered protocadherin isoforms, which have remarkable homophilic specificity for interactions between cellular surfaces. A large antiparallel dimer interface formed by the first 4 extracellular cadherin (EC) domains controls this interaction. To understand how specificity is achieved between the numerous paralogs, we used a combination of structural and computational approaches. Molecular dynamics simulations revealed that individual EC interactions are weak and undergo binding and unbinding events, but together they form a stable complex through polyvalency. Strongly evolutionarily coupled residue pairs interacted more frequently in our simulations, suggesting that sequence coevolution can inform the frequency of interaction and biochemical nature of a residue interaction. With these simulations and sequence coevolution, we generated a statistical model of interaction energy for the clustered protocadherin family that measures the contributions of all amino acid pairs at the interface. Our interaction energy model assesses specificity for all possible pairs of isoforms, recapitulating known pairings and predicting the effects of experimental changes in isoform specificity that are consistent with literature results. Our results show that sequence coevolution can be used to understand specificity determinants in a protein family and prioritize interface amino acid substitutions to reprogram specific protein–protein interactions.

The shape, elongation, division and sporulation (SEDS) proteins are a highly conserved family of transmembrane glycosyltransferases that work in concert with class B penicillin-binding proteins (bPBPs) to build the bacterial peptidoglycan cell wall1–6. How these proteins coordinate polymerization of new glycan strands with their crosslinking to the existing peptidoglycan meshwork is unclear. Here, we report the crystal structure of the prototypical SEDS protein RodA from Thermus thermophilus in complex with its cognate bPBP at 3.3 A resolution. The structure reveals a 1:1 stoichiometric complex with two extensive interaction interfaces between the proteins: one in the membrane plane and the other at the extracytoplasmic surface. When in complex with a bPBP, RodA shows an approximately 10 A shift of transmembrane helix 7 that exposes a large membrane-accessible cavity. Negative-stain electron microscopy reveals that the complex can adopt a variety of different conformations. These data define the bPBP pedestal domain as the key allosteric activator of RodA both in vitro and in vivo, explaining how a SEDS–bPBP complex can coordinate its dual enzymatic activities of peptidoglycan polymerization and crosslinking to build the cell wall. The crystal structure of the RodA–PBP2 complex from Thermus thermophilus elucidates how binding between these two proteins regulates their abilities to polymerize and crosslink peptidoglycan during bacterial cell wall synthesis.

SummaryCoevolutionary sequence analysis has become a commonly used technique for de novo prediction of the structure and function of proteins, RNA, and protein complexes. This approach requires extensive computational pipelines that integrate multiple tools, databases, and data processing steps. We present the EVcouplings framework, a fully integrated open-source application and Python package for coevolutionary analysis. The framework enables generation of sequence alignments, calculation and evaluation of evolutionary couplings (ECs), and de novo prediction of structure and mutation effects. The application has an easy to use command line interface to run workflows with user control over all analysis parameters, while the underlying modular Python package allows interactive data analysis and rapid development of new workflows. Through this multi-layered approach, the EVcouplings framework makes the full power of coevolutionary analyses available to entry-level and advanced users.Availabilityhttps://github.com/debbiemarkslab/evcouplingsContactsander.research@gmail.com, debbie@hms.harvard.edu

Summary Coevolutionary sequence analysis has become a commonly used technique for de novo prediction of the structure and function of proteins, RNA, and protein complexes. We present the EVcouplings framework, a fully integrated open-source application and Python package for coevolutionary analysis. The framework enables generation of sequence alignments, calculation and evaluation of evolutionary couplings (ECs), and de novo prediction of structure and mutation effects. The combination of an easy to use, flexible command line interface and an underlying modular Python package makes the full power of coevolutionary analyses available to entry-level and advanced users. Availability and implementation https://github.com/debbiemarkslab/evcouplings

Protocadherins (Pcdhs) are cell adhesion and signaling proteins used by neurons to develop and maintain neuronal networks, relying on trans homophilic interactions between their extracellular cadherin (EC) repeat domains. We present the structure of the antiparallel EC1-4 homodimer of human PcdhγB3, a member of the γ subfamily of clustered Pcdhs. Structure and sequence comparisons of α, β, and γ clustered Pcdh isoforms illustrate that subfamilies encode specificity in distinct ways through diversification of loop region structure and composition in EC2 and EC3, which contains isoform-specific conservation of primarily polar residues. In contrast, the EC1/EC4 interface comprises hydrophobic interactions that provide non-selective dimerization affinity. Using sequence coevolution analysis, we found evidence for a similar antiparallel EC1-4 interaction in non-clustered Pcdh families. We thus deduce that the EC1-4 antiparallel homodimer is a general interaction strategy that evolved before the divergence of these distinct protocadherin families. DOI: http://dx.doi.org/10.7554/eLife.18449.001, eLife digest As the brain develops, nerve cells or neurons connect with one another to form complex networks. These connections form between branch-like structures, called dendrites, that project from the cell body of each neuron. To prevent unneeded connections from forming, dendrites that belong to the same neuron need a way to recognize and avoid one another. A family of proteins called protocadherins supports this process of self-avoidance. Protocadherins have three main parts or domains: an extracellular domain that faces outwards away from the cell, a transmembrane domain that sits within the cell’s surface membrane and an intracellular domain that faces into the cell’s interior. There are two major groups of protocadherins – clustered and non-clustered – and the former are responsible for the self-avoidance behavior between dendrites. Clustered protocadherins in turn comprise three subfamilies, each of which consists of multiple variants with slightly different structures (known as isoforms). The particular set of protocadherin isoforms that a neuron displays on its surface distinguishes that neuron from all others, a little like a barcode. When two dendrites meet, the protocadherins in their membranes come into contact with one another. If both dendrites come from the same neuron and therefore possess identical sets of protocadherins, then all protocadherins can form two-subunit complexes containing one copy of the same isoform from each dendrite. These complexes are called homodimers and their formation acts as a signal that informs the cell that it has encountered one of its own dendrites and should therefore not establish a connection. By using X-rays to determine the structure of a crystallized protocadherin fragment down to the level of its individual atoms, Nicoludis et al. now reveal exactly how clustered protocadherins form homodimers. The results show that each protocadherin subfamily uses a slightly different type of interaction due to differences in the structure of their extracellular domains. The next challenge is to identify the signaling cascade that is triggered by the formation of clustered protocadherin homodimers, and to work out how activation of this cascade prevents a permanent connection from forming. In addition, the results of Nicoludis et al. predict that some non-clustered protocadherins form dimers with a similar architecture to that of clustered protocadherins. This possibility should also be tested experimentally. DOI: http://dx.doi.org/10.7554/eLife.18449.002

Bacteria can evolve rapidly under positive selection owing to their vast numbers, allowing their genes to diversify by adapting to different environments. We asked whether the same genes that are fast evolving in the long-term evolution experiment with Escherichia coli (LTEE) have also diversified extensively in nature. We identified ~2000 core genes shared among 60 E. coli strains. During the LTEE, core genes accumulated significantly more nonsynonymous mutations than flexible (i.e., noncore) genes. Furthermore, core genes under positive selection in the LTEE are more conserved in nature than the average core gene. In some cases, adaptive mutations appear to fine-tune protein functions, rather than merely knocking them out. The LTEE conditions are novel for E. coli, at least in relation to the long sweep of its evolution in nature. The constancy and simplicity of the environment likely favor the complete loss of some unused functions and the fine-tuning of others.Competing Interests StatementWe, the authors, declare that we have no conflicts of interest.

The bacterial genomes of Thermotoga species show evidence of significant interdomain horizontal gene transfer from the Archaea. Members of this genus acquired many genes from the Thermococcales, which grow at higher temperatures than Thermotoga species. In order to study the functional history of an interdomain horizontally acquired gene we used ancestral sequence reconstruction to examine the thermal characteristics of reconstructed ancestral proteins of the Thermotoga lineage and its archaeal donors. Several ancestral sequence reconstruction methods were used to determine the possible sequences of the ancestral Thermotoga and Archaea myo-inositol-3-phosphate synthase (MIPS). These sequences were predicted to be more thermostable than the extant proteins using an established sequence composition method. We verified these computational predictions by measuring the activities and thermostabilities of purified proteins from the Thermotoga and the Thermococcales species, and eight ancestral reconstructed proteins. We found that the ancestral proteins from both the archaeal donor and the Thermotoga most recent common ancestor recipient were more thermostable than their descendants. We show that there is a correlation between the thermostability of MIPS protein and the optimal growth temperature (OGT) of its host, which suggests that the OGT of the ancestors of these species of Archaea and the Thermotoga grew at higher OGTs than their descendants.

In 2009, James Lake introduced a new hypothesis in which reticulate phylogeny reconstruction is used to elucidate the origin of Gram-negative bacteria (Nature 460: 967–971). The presented data supported the Gram-negative bacteria originating from an ancient endosymbiosis between the Actinobacteria and Clostridia. His conclusion was based on a presence-absence analysis of protein families that divided all prokaryotes into five groups: Actinobacteria, Double Membrane bacteria (DM), Clostridia, Archaea and Bacilli. Of these five groups, the DM are by far the largest and most diverse group compared to the other groupings. While the fusion hypothesis for the origin of double membrane bacteria is enticing, we show that the signal supporting an ancient symbiosis is lost when the DM group is broken down into smaller subgroups. We conclude that the signal detected in James Lake's analysis in part results from a systematic artifact due to group size and diversity combined with low levels of horizontal gene transfer., Exobiology Program (U.S.) (Grant NNX08AQ10G), Assembling the Tree of Life (Program) (Grant DEB 0830024)

Phylogenetic reconstruction using DNA and protein sequences has allowed the reconstruction of evolutionary histories encompassing all life. We present and discuss a means to incorporate much of this rich narrative into a single model that acknowledges the discrete evolutionary units that constitute the organism. Briefly, this Rooted Net of Life genome phylogeny is constructed around an initial, well resolved and rooted tree scaffold inferred from a supermatrix of combined ribosomal genes. Extant sampled ribosomes form the leaves of the tree scaffold. These leaves, but not necessarily the deeper parts of the scaffold, can be considered to represent a genome or pan-genome, and to be associated with members of other gene families within that sequenced (pan)genome. Unrooted phylogenies of gene families containing four or more members are reconstructed and superimposed over the scaffold. Initially, reticulations are formed where incongruities between topologies exist. Given sufficient evidence, edges may then be differentiated as those representing vertical lines of inheritance within lineages and those representing horizontal genetic transfers or endosymbioses between lineages. Reviewers W. Ford Doolittle, Eric Bapteste and Robert Beiko., National Science Foundation (U.S.) (Assembling the Tree of Life DEB 0830024), United States. National Aeronautics and Space Administration (NNX08AQ10G), United States. National Aeronautics and Space Administration (NNX07AK15G), United States. National Aeronautics and Space Administration (Postdoctoral Fellowship)