Structural basis for profilin-mediated actin nucleotide exchange

Actin is a ubiquitous eukaryotic protein that is responsible for cellular scaffolding, motility, and division. The ability of actin to form a helical filament is the driving force behind these cellular activities. Formation of a filament depends on the successful exchange of actin's ADP for ATP. Mammalian profilin is a small actin binding protein that catalyzes the exchange of nucleotide and facilitates the addition of an actin monomer to a growing filament. Here, crystal structures of profilin-actin have been determined to show an actively exchanging ATP. Structural analysis shows how the binding of profilin to the barbed end of actin causes a rotation of the small domain relative to the large domain. This conformational change is propagated to the ATP site and causes a shift in nucleotide loops, which in turn causes a repositioning of Ca(2+) to its canonical position as the cleft closes around ATP. Reversal of the solvent exposure of Trp356 is also involved in cleft closure. In addition, secondary calcium binding sites were identified.     

Structural basis of ligand activation in a cyclic nucleotide regulated potassium channel

Here we describe the initial functional characterization of a cyclic nucleotide regulated ion channel from the bacterium Mesorhizobium loti and present two structures of its cyclic nucleotide binding domain, with and without cAMP. The domains are organized as dimers with the interface formed by the linker regions that connect the nucleotide binding pocket to the pore domain. Together, structural and functional data suggest the domains form two dimers on the cytoplasmic face of the channel. We propose a model for gating in which ligand binding alters the structural relationship within a dimer, directly affecting the position of the adjacent transmembrane helices.     

Structural basis for the role of LYS220 as proton donor for nucleotidyl transfer in HIV-1 reverse transcriptase

Biochemical studies by Castro et al. have recently revealed a crucial role for a general acid in the catalysis of nucleic acid transfer in distinct classes of polymerases. For HIV-RT LYS220 was identified as proton donor. This was unanticipated from a structural point of view, since in all ternary crystal structures of HIV-RT LYS220 are too distant from the active site to fulfill this role. In this work molecular dynamics simulations were used to reveal the dynamics of HIV-RT and to provide structural evidence for the role of LYS220. During a 1μs molecular dynamics simulation LYS220 migrates toward the active site and occupies several positions enabling direct and water mediated proton transfer towards pyrophosphate. A combination of quantum mechanical and molecular mechanics methods was used to validate the different modes of interaction.     

Structural basis for endosomal targeting by FYVE domains

The FYVE domain is a conserved protein motif characterized by its ability to bind with high affinity and specificity to phosphatidylinositol 3-phosphate (PI3P), a phosphoinositide highly enriched in early endosomes. The PI3P polar head group contacts specific amino acid residues that are conserved among FYVE domains. Despite full conservation of these residues, the ability of different FYVE domains to bind to endosomes in cells is highly variable. Here we show that the endosomal localization in intact cells absolutely requires structural features intrinsic to the FYVE domain in addition to the PI3P binding pocket. These features are involved in FYVE domain dimerization and in interaction with the membrane bilayer. These interactions, which are determined by non-conserved residues, are likely to be essential for the temporal and spatial control of protein associations at the membrane-cytosol interface within the endocytic pathway.     

Structural basis of FFAT motif-mediated ER targeting

The FFAT motif is a targeting signal responsible for localizing a number of proteins to the cytosolic surface of the endoplasmic reticulum (ER) and to the nuclear membrane. FFAT motifs bind to members of the highly conserved VAP protein family, which are tethered to the cytoplasmic face of the ER by a C-terminal transmembrane domain. We have solved crystal structures of the rat VAP-A MSP homology domain alone and in complex with an FFAT motif. The co-crystal structure was used to design a VAP mutant that disrupts rat and yeast VAP-FFAT interactions in vitro. The FFAT binding-defective mutant also blocked function of the VAP homolog Scs2p in yeast. Finally, overexpression of the FFAT binding-defective VAP in COS7 cells dramatically altered ER morphology. Our data establish the structural basis of FFAT-mediated ER targeting and suggest that FFAT-targeted proteins play an important role in determining ER morphology.     

Structural basis for activation of calcineurin by calmodulin

The highly conserved phosphatase calcineurin (CaN) plays vital roles in numerous processes including T-cell activation, development and function of the central nervous system, and cardiac growth. It is activated by the calcium sensor calmodulin (CaM). CaM binds to a regulatory domain (RD) within CaN, causing a conformational change that displaces an autoinhibitory domain (AID) from the active site, resulting in activation of the phosphatase. This is the same general mechanism by which CaM activates CaM-dependent protein kinases. Previously published data have hinted that the RD of CaN is intrinsically disordered. In this work, we demonstrate that the RD is unstructured and that it folds upon binding CaM, ousting the AID from the catalytic site. The RD is 95 residues long, with the AID attached to its C-terminal end and the 24-residue CaM binding region toward the N-terminal end. This is unlike the CaM-dependent protein kinases that have CaM binding sites and AIDs immediately adjacent in sequence. Our data demonstrate that not only does the CaM binding region folds but also an ～25- to 30-residue region between it and the AID folds, resulting in over half of the RD adopting α-helical structure. This appears to be the first observation of CaM inducing folding of this scale outside of its binding site on a target protein.     

Structural basis for the design of antibiotics targeting peptide deformylase

While protein synthesis in bacteria begins with a formylated methionine, the formyl group of the nascent polypeptide is removed by peptide deformylase. Since eukaryotic protein synthesis does not involve formylation and deformylation at the N-terminus, there has been increasing interest in peptide deformylase as a potential target for antibacterial chemotherapy. Toward this end and to aid in the design of effective antibiotics targeting peptide deformylase, the structures of the protein-inhibitor complexes of both the cobalt and the zinc containing Escherichia coli peptide deformylase bound to the transition-state analogue, (S)-2-O-(H-phosphonoxy)-L-caproyl-L-leucyl-p-nitroanilide (PCLNA), have been determined. The proteins for both deformylase-inhibitor complexes show basically the same fold as for the native enzyme. The PCLNA inhibitor adopts an extended conformation and fits nicely into a hydrophobic cavity located near the metal site. On the basis of these structures, guidelines for the design of high-affinity deformylase inhibitors are suggested. As our results show that the protein residues which interact with the PCLNA inhibitor are conserved over a wide variety of species, we suggest that antibiotics targeting deformylase could have wide applicability.     

Structural basis for amplifying vinculin activation by talin

Talin interactions with vinculin are essential for focal adhesions. Curiously, talin contains three noncontiguous vinculin binding sites (VBS) that can bind individually to the vinculin head (Vh) domain. Here we report the crystal structure of the human Vh.VBS1 complex, a validated model of the Vh.VBS2 structure, and biochemical studies that demonstrate that all of talin VBSs activate vinculin by provoking helical bundle conversion of the Vh domain, which displaces the vinculin tail (Vt) domain. Thus, helical bundle conversion is a structurally conserved response in talin-vinculin interactions. Furthermore, talin VBSs bind to Vh in a mutually exclusive manner but do differ in their affinity for Vh and in their ability to displace Vt, suggesting that the strengths of these interactions could lead to differences in signaling outcome. These findings support a model in which talin binds to and activates multiple vinculin molecules to provoke rapid reorganization of the actin cytoskeleton.