Plexins are the first known transmembrane receptors that interact directly with small GTPases. On binding to certain Rho family GTPases, the receptor regulates the remodeling of the actin cytoskeleton and alters cell movement in response to semaphorin guidance cues. In a joint solution NMR spectroscopy and x-ray crystallographic study, we characterize a 120-residue cytoplasmic independent folding domain of plexin-B1 that directly binds three Rho family GTPases, Rac1, Rnd1, and RhoD. The NMR data show that, surprisingly, the Cdc42/Rac interactive binding-like motif of plexin-B1 is not involved in this interaction. Instead, all three GTPases interact with the same region, -strands 3 and 4 and a short ␣-helical segment of the plexin domain. The 2.0 Å resolution x-ray structure shows that these segments are brought together by the tertiary structure of the ubiquitin-like fold. In the crystal, the protein is dimerized with C2 symmetry through a four-stranded antiparallel -sheet that is formed outside the fold by a long loop between the monomers. This region is adjacent to the GTPase binding motifs identified by NMR. Destabilization of the dimer in solution by binding of any one of the three GTPases suggests a model for receptor regulation that involves bidirectional signaling. The model implies a multifunctional role for the GTPase-plexin interaction that includes conformational change and a localization of active receptors in the signaling mechanism.Members of the plexin family of transmembrane receptors have important functions in guiding axon growth in the developing nervous system (1-3). Plexins also function in several developmental processes such as cardiovascular development and angiogenesis (4 -8), in the invasive growth of epithelial cells (9), and in the immune system (10). The extracellular part of plexin shares significant homology with that of the hepatocyte growth factor receptor as well as with semaphorins, the principal family of ligands for plexins. The cytoplasmic region of plexins is also well conserved (11). Two segments with homology to Ras GTPase-activating proteins (GAPs) 5 are linked by a region that has been identified as the location for the binding of several Rho family GTPases.Plexins are unique in that they are the first documented example of a transmembrane receptor that interacts directly with small GTPases. Most A-and B-family plexins bind activated Rac1 and Rnd1 (12-15), Rho family GTPases that are known regulators of cytoskeletal dynamics and cell adhesion (16). Plexin-A1 has also been shown to bind active RhoD (17), another Rho family GTPase involved in actin remodeling and endosomal dynamics and possibly in receptor down-regulation (18). The role of these plexin-Rho GTPase interactions has remained unclear, however, as have the characteristics of the binding region. Does the Rho GTPase binding segment provide a GTPase regulatory property, functioning akin to a guanine nucleotide dissociation inhibitor by sequestering certain Rho family GTPases, or does it act as an effector pro...
Age-related cataract is a major cause of blindness worldwide, and cortical cataract is the second most prevalent type of age-related cataract. Although a significant fraction of age-related cataract is heritable, the genetic basis remains to be elucidated. We report that homozygous deletion of Epha2 in two independent strains of mice developed progressive cortical cataract. Retroillumination revealed development of cortical vacuoles at one month of age; visible cataract appeared around three months, which progressed to mature cataract by six months. EPHA2 protein expression in the lens is spatially and temporally regulated. It is low in anterior epithelial cells, upregulated as the cells enter differentiation at the equator, strongly expressed in the cortical fiber cells, but absent in the nuclei. Deletion of Epha2 caused a significant increase in the expression of HSP25 (murine homologue of human HSP27) before the onset of cataract. The overexpressed HSP25 was in an underphosphorylated form, indicating excessive cellular stress and protein misfolding. The orthologous human EPHA2 gene on chromosome 1p36 was tested in three independent worldwide Caucasian populations for allelic association with cortical cataract. Common variants in EPHA2 were found that showed significant association with cortical cataract, and rs6678616 was the most significant in meta-analyses. In addition, we sequenced exons of EPHA2 in linked families and identified a new missense mutation, Arg721Gln, in the protein kinase domain that significantly alters EPHA2 functions in cellular and biochemical assays. Thus, converging evidence from humans and mice suggests that EPHA2 is important in maintaining lens clarity with age.
The sterile alpha motif (SAM) for protein-protein interactions is encountered in over 200 proteins, but the structural bases for its interactions is just becoming clear. Here we solved the structure of the EphA2-SHIP2 SAM:SAM heterodimeric complex by use of NMR restraints from chemical shift perturbations, NOE and RDC experiments. Specific contacts between the protein surfaces differ significantly from a previous model and from other SAM:SAM complexes. Molecular dynamics and docking simulations indicate fluctuations in the complex towards alternate, higher energy conformations. The interface suggests that EphA family members bind to SHIP2 SAM whereas EphB members may not; correspondingly we demonstrate binding of EphA1 but not of EphB2 to SHIP2 SAM. A variant of EphB2 SAM was designed that binds SHIP2. Functional characterization of a mutant EphA2 compromised in SHIP2 binding reveals two previously unrecognized functions of SHIP2 in suppressing ligand-induced activation of EphA2 and in promoting chemotactic cell migration in coordination with the receptor.
Although they represent attractive therapeutic targets, caspases have so far proven recalcitrant to the development of drugs targeting the active site. Allosteric modulation of caspase activity is an alternate strategy that potentially avoids the need for anionic and electrophilic functionality present in most active-site inhibitors. Caspase-6 has been implicated in neurodegenerative disease, including Huntington's and Alzheimer's diseases. Herein we describe a fragment-based lead discovery effort focused on caspase-6 in its active and zymogen forms. Fragments were identified for procaspase-6 using surface plasmon resonance methods and subsequently shown by X-ray crystallography to bind a putative allosteric site at the dimer interface. A fragment-merging strategy was employed to produce nanomolar-affinity ligands that contact residues in the L2 loop at the dimer interface, significantly stabilizing procaspase-6. Because rearrangement of the L2 loop is required for caspase-6 activation, our results suggest a strategy for the allosteric control of caspase activation with drug-like small molecules.
Inhibition of caspase-6 is a potential therapeutic strategy for some neurodegenerative diseases, but it has been difficult to develop selective inhibitors against caspases. We report the discovery and characterization of a potent inhibitor of caspase-6 that acts by an uncompetitive binding mode that is an unprecedented mechanism of inhibition against this target class. Biochemical assays demonstrate that, while exquisitely selective for caspase-6 over caspase-3 and -7, the compound’s inhibitory activity is also dependent on the amino acid sequence and P1’ character of the peptide substrate. The crystal structure of the ternary complex of caspase-6, substrate-mimetic and an 11 nM inhibitor reveals the molecular basis of inhibition. The general strategy to develop uncompetitive inhibitors together with the unique mechanism described herein provides a rationale for engineering caspase selectivity.
Although poorly understood, the properties of the denatured state ensemble are critical to the thermodynamics and the kinetics of protein folding. The most relevant conformations to cellular protein folding are the ones populated under physiological conditions. To avoid the problem of low expression that is seen with unstable variants, we used methionine oxidation to destabilize monomeric l repressor and predominantly populate the denatured state under nondenaturing buffer conditions. The denatured ensemble populated under these conditions comprises conformations that are compact. Analytical ultracentrifugation sedimentation velocity experiments indicate a small increase in Stokes radius over that of the native state. A significant degree of a-helical structure in these conformations is detected by far-UV circular dichroism, and some tertiary interactions are suggested by near-UV circular dichroism. The characteristics of the denatured state populated by methionine oxidation in nondenaturing buffer are very different from those found in chemical denaturant.Keywords: repressor proteins; denatured state ensemble; methionine oxidation; hydrodynamic radius; heteronuclear NMR; protein structure/folding; NMR spectroscopy; new methods; circular dichroism Supplemental material: see www.proteinscience.org Many small single-domain proteins required for cellular function fold reversibly without the aid of chaperones (Jackson 1998). The starting point for reversible folding is the denatured state within the cell. A detailed description of this denatured state is necessary to understand the thermodynamics and kinetics of protein folding (Shortle 2002). For example, residual structure in the denatured state has been shown to differentially affect the thermodynamic stability of RNase H from Escherichia coli and Thermus thermophilus (Robic et al. 2003). The structural properties of the denatured state also provide clues to early events in protein folding (Shortle 2002). While it is impossible to duplicate the cellular environment in vitro, the denatured state under physiological conditions is most relevant. Traditional methods used to populate the denatured state, including high concentrations of chemical denaturants, high temperatures, and low pH are poor models for denatured proteins in vivo because of the difference in solvent conditions compared to cellular conditions. In this paper we present evidence that the denatured state of the monomeric l repressor in solvent conditions that are more like the cellular environment is significantly different from the one populated by addition of chemical denaturant.Amino acid substitutions can be used to locally perturb the energy of a protein, thereby shifting the folding equilibrium to favor the denatured state under conditions that normally favor the folded native state. However, Reprint requests to: Terrence G. Oas, Department of Biochemistry, 436 Nanaline Duke Building, Box 3711, Duke University Medical Center, Durham, NC 27710, USA; e-mail: oas@duke.edu; fax: (919) 681-88...
Oxidizing two native methionine residues predominantly populates the denatured state of monomeric lambda repressor (MetO-lambdaLS) under nondenaturing conditions. NMR was used to characterize the secondary structure and dynamics of MetO-lambdaLS in standard phosphate buffer. 13Calpha and 1Halpha chemical shift indices reveal a region of significant helicity between residues 9 and 29. This helical content is further supported by the observation of medium-range amide NOEs. The remaining residues do not exhibit significant helicity as determined by NMR. We determined 15N relaxation parameters for 64 of 85 residues at 600 and 800 MHz. There are two distinct regions of reduced flexibility, residues 8-32 in the N-terminal third and residues 50-83 in the C-terminal third. The middle third, residues 33-50, has greater flexibility. We have analyzed the amplitude of the backbone motions in terms of the physical properties of the amino acids and conclude that conformational restriction of the backbone MetO-lambdaLS is due to nascent helix formation in the region corresponding to native helix 1. The bulkiness of amino acid residues in the C-terminal third leads to the potential for hydrophobic interactions, which is suggested by chemical exchange detected by the difference in spectral density J(0) at the two static magnetic fields. The more flexible middle region is the result of a predominance of small side chains in this region.
The inside cover picture shows small molecules tailored for an allosteric site in procaspase-6. The combination of biophysical fragment screening, structure-and computation-aided design, and chemical synthesis enabled the discovery of multiple nanomolar-affinity ligands for this new site. The compounds significantly stabilize the protein, suggesting new avenues for controlling caspase activity and/or activation by allosteric mechanisms. For more details, see the Communication by Jeremy Murray, Adam R. Renslo et al. on p. 73 ff.
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