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.

Netrin Synergizes Signaling and Adhesion through DCC.

Netrin is a prototypical axon guidance cue. Structural studies have revealed how netrin interacts with the deleted in colorectal cancer (DCC) receptor, other receptors, and co-factors for signaling. Recently, genetic studies suggested that netrin is involved in neuronal haptotaxis, which requires a reversible adhesion process. Structural data indicate that netrin can also mediate trans-adhesion between apposing cells decorated with its receptors on the condition that the auxiliary guidance cue draxin is present. Here, we propose that netrin is involved in conditional adhesion, a reversible and localized process that can contribute to cell adhesion and migration. We suggest that netrin-mediated adhesion and signaling are linked, and that local environmental factors in the ventricular zone, the floor plate, or other tissues coordinate its function.

Roles of glycosaminoglycans as regulators of ligand/receptor complexes.

Glycosaminoglycans (GAGs) play a widespread role in embryonic development, as deletion of enzymes that contribute to GAG synthesis lead to deficiencies in cell migration and tissue modelling. Despite the biochemical and structural characterization of individual protein/GAG interactions, there is no concept available that links the molecular mechanisms of GAG/protein engagements to tissue development. Here, we focus on the role of GAG polymers in mediating interactions between cell surface receptors and their ligands. We categorize several switches that lead to ligand activation, inhibition, selection and addition, based on recent structural studies of select receptor/ligand complexes. Based on these principles, we propose that individual GAG polymers may affect several receptor pathways in parallel, orchestrating a cellular response to an environmental cue. We believe that it is worthwhile to study the role of GAGs as molecular switches, as this may lead to novel drug candidates to target processes such as angiogenesis, neuroregeneration and tumour metastasis.

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 ß-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.

Conserved, disordered C terminus of DnaK enhances cellular survival upon stress and DnaK in vitro chaperone activity.

The 70-kDa heat shock proteins (Hsp70s) function as molecular chaperones through the allosteric coupling of their nucleotide- and substrate-binding domains, the structures of which are highly conserved. In contrast, the roles of the poorly structured, variable length C-terminal regions present on Hsp70s remain unclear. In many eukaryotic Hsp70s, the extreme C-terminal EEVD tetrapeptide sequence associates with co-chaperones via binding to tetratricopeptide repeat domains. It is not known whether this is the only function for this region in eukaryotic Hsp70s and what roles this region performs in Hsp70s that do not form complexes with tetratricopeptide repeat domains. We compared C-terminal sequences of 730 Hsp70 family members and identified a novel conservation pattern in a diverse subset of 165 bacterial and organellar Hsp70s. Mutation of conserved C-terminal sequence in DnaK, the predominant Hsp70 in Escherichia coli, results in significant impairment of its protein refolding activity in vitro without affecting interdomain allostery, interaction with co-chaperones DnaJ and GrpE, or the binding of a peptide substrate, defying classical explanations for the chaperoning mechanism of Hsp70. Moreover, mutation of specific conserved sites within the DnaK C terminus reduces the capacity of the cell to withstand stresses on protein folding caused by elevated temperature or the absence of other chaperones. These features of the C-terminal region support a model in which it acts as a disordered tether linked to a conserved, weak substrate-binding motif and that this enhances chaperone function by transiently interacting with folding clients.

An interdomain sector mediating allostery in Hsp70 molecular chaperones.

Allosteric coupling between protein domains is fundamental to many cellular processes. For example, Hsp70 molecular chaperones use ATP binding by their actin-like N-terminal ATPase domain to control substrate interactions in their C-terminal substrate-binding domain, a reaction that is critical for protein folding in cells. Here, we generalize the statistical coupling analysis to simultaneously evaluate co-evolution between protein residues and functional divergence between sequences in protein sub-families. Applying this method in the Hsp70/110 protein family, we identify a sparse but structurally contiguous group of co-evolving residues called a 'sector', which is an attribute of the allosteric Hsp70 sub-family that links the functional sites of the two domains across a specific interdomain interface. Mutagenesis of Escherichia coli DnaK supports the conclusion that this interdomain sector underlies the allosteric coupling in this protein family. The identification of the Hsp70 sector provides a basis for further experiments to understand the mechanism of allostery and introduces the idea that cooperativity between interacting proteins or protein domains can be mediated by shared sectors.

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 (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 semilabeled 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.

Sending signals dynamically.

Proteins mediate transmission of signals along intercellular and intracellular pathways and between the exterior and the interior of a cell. The dynamic properties of signaling proteins are crucial to their functions. We discuss emerging paradigms for the role of protein dynamics in signaling. A central tenet is that proteins fluctuate among many states on evolutionarily selected energy landscapes. Upstream signals remodel this landscape, causing signaling proteins to transmit information to downstream partners. New methods provide insight into the dynamic properties of signaling proteins at the atomic scale. The next stages in the signaling hierarchy-how multiple signals are integrated and how cellular signaling pathways are organized in space and time-present exciting challenges for the future, requiring bold multidisciplinary approaches.

Evolutionary coupling of structural and functional sequence information in the intracellular lipid-binding protein family.

We have mined the evolutionary record for the large family of intracellular lipid-binding proteins (iLBPs) by calculating the statistical coupling of residue variations in a multiple sequence alignment using methods developed by Ranganathan and coworkers (Lockless and Ranganathan, Science 1999:286;295-299). The 213 sequences analyzed have a wide range of ligand-binding functions as well as highly divergent phylogenetic origins, assuring broad sampling of sequence space. Emerging from this analysis were two major clusters of coupled residues, which when mapped onto the structure of a representative iLBP under study in our laboratory, cellular retinoic-acid binding protein I, are largely contiguous and provide useful points of comparison to available data for the folding of this protein. One cluster comprises a predominantly hydrophobic core away from the ligand-binding site and likely represents key structural information for the iLBP fold. The other cluster includes the portal region where ligand enters its binding site, regions of the ligand-binding cavity, and the region where the 10-stranded beta-barrel characteristic of this family closes (between strands 1' and 10). Linkages between these two clusters suggest that evolutionary pressures on this family constrain structural and functional sequence information in an interdependent fashion. The necessity of the structure to wrap around a hydrophobic ligand confounds the typical sequestration of hydrophobic side chains. Additionally, ligand entry and exit require these structures to have a capacity for specific conformational change during binding and release. We conclude that an essential and structurally apparent separation of local and global sequence information is conserved throughout the iLBP family.

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.