Computational de novo design of a four-helix bundle protein—DND_4HB

The de novo design of proteins is a rigorous test of our understanding of the key determinants of protein structure. The helix bundle is an interesting de novo design model system due to the diverse topologies that can be generated from a few simple α-helices. Previously, noncomputational studies demonstrated that connecting amphipathic helices together with short loops can sometimes generate helix bundle proteins, regardless of the bundle''s exact sequence. However, using such methods, the precise positions of helices and side chains cannot be predetermined. Since protein function depends on exact positioning of residues, we examined if sequence design tools in the program Rosetta could be used to design a four-helix bundle with a predetermined structure. Helix position was specified using a folding procedure that constrained the design model to a defined topology, and iterative rounds of rotamer-based sequence design and backbone refinement were used to identify a low energy sequence for characterization. The designed protein, DND_4HB, unfolds cooperatively (T_m >90°C) and a NMR solution structure shows that it adopts the target helical bundle topology. Helices 2, 3, and 4 agree very closely with the design model (backbone RMSD = 1.11 Å) and >90% of the core side chain χ1 and χ2 angles are correctly predicted. Helix 1 lies in the target groove against the other helices, but is displaced 3 Å along the bundle axis. This result highlights the potential of computational design to create bundles with atomic-level precision, but also points at remaining challenges for achieving specific positioning between amphipathic helices.     

Structural basis of the collagen-binding mode of discoidin domain receptor 2

Discoidin domain receptor (DDR) is a cell-surface receptor tyrosine kinase activated by the binding of its discoidin (DS) domain to fibrillar collagen. Here, we have determined the NMR structure of the DS domain in DDR2 (DDR2-DS domain), and identified the binding site to fibrillar collagen by transferred cross-saturation experiments. The DDR2-DS domain structure adopts a distorted jellyroll fold, consisting of eight beta-strands. The collagen-binding site is formed at the interloop trench, consisting of charged residues surrounded by hydrophobic residues. The surface profile of the collagen-binding site suggests that the DDR2-DS domain recognizes specific sites on fibrillar collagen. This study provides a molecular basis for the collagen-binding mode of the DDR2-DS domain.     

Solution structure of choline binding protein A, the major adhesin of Streptococcus pneumoniae

Streptococcus pneumoniae (pneumococcus) remains a significant health threat worldwide, especially to the young and old. While some of the biomolecules involved in pneumococcal pathogenesis are known and understood in mechanistic terms, little is known about the molecular details of bacterium/host interactions. We report here the solution structure of the 'repeated' adhesion domains (domains R1 and R2) of the principal pneumococcal adhesin, choline binding protein A (CbpA). Further, we provide insights into the mechanism by which CbpA binds its human receptor, polymeric immunoglobulin receptor (pIgR). The R domains, comprised of 12 imperfect copies of the leucine zipper heptad motif, adopt a unique 3-alpha-helix, raft-like structure. Each pair of alpha-helices is antiparallel and conserved residues in the loop between Helices 1 and 2 exhibit a novel 'tyrosine fork' structure that is involved in binding pIgR. This and other structural features that we show are conserved in most pneumococcal strains appear to generally play an important role in bacterial adhesion to pIgR. Interestingly, pneumococcus is the only bacterium known to adhere to and invade human cells by binding to pIgR.