No abstract

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

Abundant and essential motifs, such as phosphate-binding loops (P-loops), are presumed to be the seeds of modern enzymes. The Walker-A P-loop is absolutely essential in modern NTPase enzymes, in mediating binding, and transfer of the terminal phosphate groups of NTPs. However, NTPase function depends on many additional active-site residues placed throughout the protein’s scaffold. Can motifs such as P-loops confer function in a simpler context? We applied a phylogenetic analysis that yielded a sequence logo of the putative ancestral Walker-A P-loop element: a β-strand connected to an α-helix via the P-loop. Computational design incorporated this element into de novo designed β-α repeat proteins with relatively few sequence modifications. We obtained soluble, stable proteins that unlike modern P-loop NTPases bound ATP in a magnesium-independent manner. Foremost, these simple P-loop proteins avidly bound polynucleotides, RNA, and single-strand DNA, and mutations in the P-loop’s key residues abolished binding. Binding appears to be facilitated by the structural plasticity of these proteins, including quaternary structure polymorphism that promotes a combined action of multiple P-loops. Accordingly, oligomerization enabled a 55-aa protein carrying a single P-loop to confer avid polynucleotide binding. Overall, our results show that the P-loop Walker-A motif can be implemented in small and simple β-α repeat proteins, primarily as a polynucleotide binding motif.

SUMMARY Protein flexibility ranges from simple hinge movements to functional disorder. Around half of all human proteins contain apparently disordered regions, with little 3D or functional information, and many of these proteins are associated with disease. Building on the evolutionary couplings approach previously successful in predicting 3D states of ordered proteins and RNA, we developed a method to predict the potential for ordered states for all apparently disordered proteins with sufficiently rich evolutionary information. The approach is highly accurate (79%) for residue interactions as tested in over 60 known disordered regions captured in a bound or specific condition. Assessing the potential for structure of over 1000 apparently-disordered regions of human proteins reveals a continuum of structural order with at least 50% with clear propensity for three- or two-dimensional states. Co-evolutionary constraints reveal hitherto unseen structure of functional importance in apparently disordered proteins.