De Novo Evolutionary Emergence of a Symmetrical Protein Is Shaped by Folding Constraints.

Molecular evolution has focused on the divergence of molecular functions, yet we know little about how structurally distinct protein folds emerge de novo. We characterized the evolutionary trajectories and selection forces underlying emergence of β-propeller proteins, a globular and symmetric fold group with diverse functions. The identification of short propeller-like motifs (<50 amino acids) in natural genomes indicated that they expanded via tandem duplications to form extant propellers. We phylogenetically reconstructed 47-residue ancestral motifs that form five-bladed lectin propellers via oligomeric assembly. We demonstrate a functional trajectory of tandem duplications of these motifs leading to monomeric lectins. Foldability, i.e., higher efficiency of folding, was the main parameter leading to improved functionality along the entire evolutionary trajectory. However, folding constraints changed along the trajectory: initially, conflicts between monomer folding and oligomer assembly dominated, whereas subsequently, upon tandem duplication, tradeoffs between monomer stability and foldability took precedence.We thank Michael Gurevitz (Tel Aviv University), John Finnerty (Boston University) and Adam Reitzel (Woodshole Oceanographic Institute) for providing N. vectensis cDNA, and Joseph Rogers (University of Cambridge) for discussion and assistance. We thank Liam Longo, Ron Milo and Balaji Santhanam for insightful comments on this manuscript. This work was supported by the Israel Science Foundation grant 980/14 (DST), the Weizmann - UK Joint Research Program (DST and JC), the Weizmann Koshland and Dean of Faculty fellowships (RGS) and an EMBO short-term fellowship (RGS). JC is a Wellcome Trust Fellow (WT 095195).This is the final version of the article. It first appeared from Elsevier via http://dx.doi.org/10.1016/j.cell.2015.12.02

An interdomain sector mediating allostery in Hsp70 molecular chaperones

The Hsp70 family of molecular chaperones provides a well defined and experimentally powerful model system for understanding allosteric coupling between different protein domains.New extensions to the statistical coupling analysis (SCA) method permit identification of a group of co-evolving amino-acid positions—a sector—in the Hsp70 that is associated with allosteric function.Literature-based and new experimental studies support the notion that the protein sector identified through SCA underlies the allosteric mechanism of Hsp70.This work extends the concept of protein sectors by showing that two non-homologous protein domains can share a single sector when the underlying biological function is defined by the coupled activity of the two domains

Activation of PKA via asymmetric allosteric coupling of structurally conserved cyclic nucleotide binding domains

Cyclic nucleotide-binding (CNB) domains allosterically regulate the activity of proteins with diverse functions, but the mechanisms that enable the cyclic nucleotide-binding signal to regulate distant domains are not well understood. Here we use optical tweezers and molecular dynamics to dissect changes in folding energy landscape associated with cAMP-binding signals transduced between the two CNB domains of protein kinase A (PKA). We find that the response of the energy landscape upon cAMP binding is domain specific, resulting in unique but mutually coordinated tasks: one CNB domain initiates cAMP binding and cooperativity, whereas the other triggers inter-domain interactions that promote the active conformation. Inter-domain interactions occur in a stepwise manner, beginning in intermediate-liganded states between apo and cAMP-bound domains. Moreover, we identify a cAMP-responsive switch, the N3A motif, whose conformation and stability depend on cAMP occupancy. This switch serves as a signaling hub, amplifying cAMP-binding signals during PKA activation

Components of a Protein Machine: Allosteric Domain Assembly and a Disordered C-terminus Enable the Chaperone Functions of Hsp70

Hsp70 molecular chaperones protect proteins from aggregation, assist in their native structure formation, and regulate stress responses in the cell. A mechanistic understanding of Hsp70 function will be necessary to explain its physiological roles and guide the therapeutic modulation of various disease states. To this end, several fundamental features of the Hsp70 structure-function relationship are investigated. The central component of Hsp70 chaperone function is its capacity for allosteric signaling between structural domains and tunable binding of misfolded protein substrates. In order to identify a cooperative network of sites that mediates interdomain allostery within Hsp70, a mutational correlation analysis is performed using genetic data. Evolutionarily correlations that describe an allosteric network are validated by examining roles for implicated sites in cellular fitness and molecular function. In a second component of the Hsp70 molecular mechanism, a novel function is discovered for the disordered C-terminal tail. This region of the protein enhances the refolding efficiency of substrate proteins independently of interdomain allostery and is required in the cell upon depletion of compensatory chaperones, suggesting a previously undescribed mode of chaperone action. Finally, experiments are initiated to assess the dynamic assembly of Hsp70 domains in various allosteric states and how domain orientations may be guided through interaction with partner co-chaperone proteins

Finding the Fittest Fold: Using the Evolutionary Record to Design New Proteins

For many years, the holy grail of protein engineering has been the design of artificial amino acid sequences that fold into stable proteins with desired functions. In the current issue of Nature, two papers from the Ranganathan group (Russ et al., 2005; Socolich et al., 2005) report remarkable success in the design of artificial WW domains. Their method, termed statistical coupling analysis (Lockless and Ranganathan, 1999), does not use structural or physicochemical information but instead extracts information about essential patterns of amino acids from the evolutionary record

Segmental isotopic labeling of the Hsp70 molecular chaperone DnaK using expressed protein ligation

Introducing biophysical labels into specific regions of large and dynamic multidomain proteins greatly facilitates mechanistic analysis. Ligation of expressed domains that are labeled in a desired manner before assembly of the intact molecular machine provides such a strategy. We have elaborated an experimental route using expressed protein ligation (EPL) to create an Hsp70 molecular chaperone (in this case the E. coli Hsp70, DnaK) where only one of the two constituent domains was labeled, in this case with NMR active isotopes, allowing visualization of the single domain in the context of the two domain protein. Several technical obstacles were overcome, including choice of site for ligation with retention of function, optimization of ligation yield, and purification from unreacted domains. Ligated semi-labeled DnaK was successfully produced with a Cys residue at position 383, and the ligated product harboring the Cys mutation was confirmed to be functional and identical to an expressed Cys-containing two-domain construct. The NMR spectrum of the segmentally labeled protein was considerably simplified, enabling unequivocal assignment and enhanced analysis of dynamics, as a prelude to exploring the energy landscape for allostery in the Hsp70 family

Structure of unliganded membrane-proximal domains FN4-FN5-FN6 of DCC

Ministry of Education of China; National Natural Science Foundation of China (NSFC) [81425009]; NSFC [31630028, 91632305]; NIH [HL103526]; Peking-Tsinghua Center for Life Sciences; European Research Council (ERC) under a Horizon MSCA-IF [702346]SCI(E)LETTER9701-705

Structural insights into the formation of repulsive netrin guidance complexes

Netrins dictate attractive and repulsive responses during axon growth and cell migration, where the presence of the receptor Uncoordinated-5 (UNC-5) on target cells results in repulsion. Here, we showed that UNC-5 is a heparin-binding protein, determined its structure bound to a heparin fragment, and could modulate UNC-5–heparin affinity using a directed evolution platform or structure-based rational design. We demonstrated that UNC-5 and UNC-6/netrin form a large, stable, and rigid complex in the presence of heparin, and heparin and UNC-5 exclude the attractive UNC-40/DCC receptor from binding to UNC-6/netrin to a large extent. Caenorhabditis elegans with a heparin-binding–deficient UNC-5 fail to establish proper gonad morphology due to abrogated cell migration, which relies on repulsive UNC-5 signaling in response to UNC-6. Combining UNC-5 mutations targeting heparin and UNC-6/netrin contacts results in complete cell migration and axon guidance defects. Our findings establish repulsive netrin responses to be mediated through a glycosaminoglycan-regulated macromolecular complex