Biomedical subjects

Wendell A Lim

Publications and source records attributed to Wendell A Lim.

At least 19 recordsLinked to original sources

PubMed2007

Multiple WASP-interacting protein recognition motifs are required for a functional interaction with N-WASP.

The WASP-interacting protein (WIP) targets WASP/WAVE proteins through a constitutive interaction with an amino-terminal enabled/VASP homology (EVH1) domain. Parallel investigations had previously identified two distinct N-WASP binding motifs corresponding to WIP residues 451-461 and 461-485, and we determined the structure of a complex between WIP-(461-485) and the N-WASP EVH1 domain (Volkman, B. F., Prehoda, K. E., Scott, J. A., Peterson, F. C., and Lim, W. A. (2002) Cell 111, 565-576). The present results show that, when combined, the WIP-(451-485) sequence wraps further around the EVH1 domain, extending the interface observed previously. Specific contacts with three WIP epitopes corresponded to regions of high sequence conservation in the verprolin family. A central polyproline motif occupied the canonical binding site but in a reversed orientation relative to other EVH1 complexes. This interaction was augmented in the amino- and carboxyl-terminal directions by additional hydrophobic contacts involving WIP residues 454-459 and 475-478, respectively. Disruption of any of the three WIP epitopes reduced N-WASP binding in cells, demonstrating a functional requirement for the entire binding domain, which is significantly longer than the polyproline motifs recognized by other EVH1 domains.

Amino Acid Motifs↗

PubMed2006

Docking interactions in protein kinase and phosphatase networks.

To achieve high biological specificity, protein kinases and phosphatases often recognize their targets through interactions that occur outside of the active site. Although the role of modular protein-protein interaction domains in kinase and phosphatase signaling has been well characterized, it is becoming clear that many kinases and phosphatases utilize docking interactions - recognition of a short peptide motif in target partners by a groove on the catalytic domain that is separate from the active site. Docking is particularly prevalent in serine/threonine kinases and phosphatases, and is a versatile organizational tool for building complex signaling networks; it confers a high degree of specificity and, in some cases, allosteric regulation.

Allosteric Regulation↗

PubMed2006

The Ste5 scaffold allosterically modulates signaling output of the yeast mating pathway.

Scaffold proteins organize signaling proteins into pathways and are often viewed as passive assembly platforms. We found that the Ste5 scaffold has a more active role in the yeast mating pathway: A fragment of Ste5 allosterically activated autophosphorylation of the mitogen-activated protein kinase Fus3. The resulting form of Fus3 is partially active-it is phosphorylated on only one of two key residues in the activation loop. Unexpectedly, at a systems level, autoactivated Fus3 appears to have a negative regulatory role, promoting Ste5 phosphorylation and a decrease in pathway transcriptional output. Thus, scaffolds not only direct basic pathway connectivity but can precisely tune quantitative pathway input-output properties.

Adaptor Proteins, Signal Transducing↗

PubMed2006

Beta strand peptidomimetics as potent PDZ domain ligands.

The search for general strategies for inhibiting protein-protein interactions has been stimulated by recognition of the key role they play in virtually every process of living systems. Multiprotein complex assembly and localization by PDZ domain-containing proteins exemplify processes critical to cell physiology and function that are mediated by beta strand association. Here we describe the development of substituted "@-tides," protease-resistant peptidomimetics incorporating conformationally restricted amino acid surrogates that reproduce the hydrogen-bonding pattern and side-chain functionality of a beta strand. The synthetic flexibility and generality of the substituted @-tide design was demonstrated by the synthesis of a panel of ligands for the alpha1-syntrophin PDZ domain. The rational design of a small molecule of unprecedented affinity for the PDZ domain suggests that these peptidomimetics may provide a general method for inhibiting protein-protein interactions involving extended peptide chains.

Biomimetics↗

PubMed2006

Domains, motifs, and scaffolds: the role of modular interactions in the evolution and wiring of cell signaling circuits.

Living cells display complex signal processing behaviors, many of which are mediated by networks of proteins specialized for signal transduction. Here we focus on the question of how the remarkably diverse array of eukaryotic signaling circuits may have evolved. Many of the mechanisms that connect signaling proteins into networks are highly modular: The core catalytic activity of a signaling protein is physically and functionally separable from molecular domains or motifs that determine its linkage to both inputs and outputs. This high degree of modularity may make these systems more evolvable-in principle, novel circuits, and therefore highly innovative regulatory behaviors, can arise from relatively simple genetic events such as recombination, deletion, or insertion. In support of this hypothesis, recent studies show that such modular systems can be exploited to engineer nonnatural signaling proteins and pathways with novel behavior.

Animals↗

PubMed2005

The role of docking interactions in mediating signaling input, output, and discrimination in the yeast MAPK network.

Cells use a network of mitogen-activated protein kinases (MAPKs) to coordinate responses to diverse extracellular signals. Here, we examine the role of docking interactions in determining connectivity of the yeast MAPKs Fus3 and Kss1. These closely related kinases are activated by the common upstream MAPK kinase Ste7 yet generate distinct output responses, mating and filamentous growth, respectively. We find that docking interactions are necessary for communication with the kinases and that they can encode subtle differences in pathway-specific input and output. The cell cycle arrest mediator Far1, a mating-specific substrate, has a docking motif that selectively binds Fus3. In contrast, the shared partner Ste7 has a promiscuous motif that binds both Fus3 and Kss1. Structural analysis reveals that Fus3 interacts with specific and promiscuous peptides in conformationally distinct modes. Induced fit recognition may allow docking peptides to achieve discrimination by exploiting subtle differences in kinase flexibility.

Amino Acid Sequence↗

PubMed2005

A general model for preferential hetero-oligomerization of LIN-2/7 domains: mechanism underlying directed assembly of supramolecular signaling complexes.

LIN-2/7 (L27) domains are protein interaction modules that preferentially hetero-oligomerize, a property critical for their function in directing specific assembly of supramolecular signaling complexes at synapses and other polarized cell-cell junctions. We have solved the solution structure of the heterodimer composed of the L27 domains from LIN-2 and LIN-7. Comparison of this structure with other L27 domain structures has allowed us to formulate a general model for why most L27 domains form an obligate heterodimer complex. L27 domains can be divided in two types (A and B), with each heterodimer comprising an A/B pair. We have identified two keystone positions that play a central role in discrimination. The residues at these positions are energetically acceptable in the context of an A/B heterodimer, but would lead to packing defects or electrostatic repulsion in the context of A/A and B/B homodimers. As predicted by the model, mutations of keystone residues stabilize normally strongly disfavored homodimers. Thus, L27 domains are specifically optimized to avoid homodimeric interactions.

Adaptor Proteins, Signal Transducing↗

PubMed2005

A polybasic motif allows N-WASP to act as a sensor of PIP(2) density.

Phosphatidylinositol 4,5-bisphosphate (PIP(2)) activates the actin regulatory protein N-WASP by binding to a short polybasic region involved in N-WASP autoinhibition. Here, we show that unlike canonical lipid binding modules, such as PH domains, this polybasic motif binds PIP(2) in a multivalent, cooperative manner. As a result, PIP(2) activation of N-WASP-mediated actin polymerization in vitro and in extracts is ultrasensitive: above a certain threshold, N-WASP responds in a switch-like manner to a small increase in the density of PIP(2) (Hill coefficient n(H) = approximately 20). We show that the sharpness of the PIP(2) activation threshold can be tuned by varying the length of the polybasic motif. This sharp activation threshold may help suppress N-WASP activation by quiescent PIP(2) levels yet leave it poised for activation upon subtle, signaling-induced perturbations in PIP(2) distribution.

Actins↗

PubMed2004

Sho1 and Pbs2 act as coscaffolds linking components in the yeast high osmolarity MAP kinase pathway.

Scaffold proteins mediate efficient and specific signaling in several mitogen-activated protein (MAP) kinase cascades. In the yeast high osmolarity response pathway, the MAP kinase kinase Pbs2 is thought to function as a scaffold, since it binds the osmosensor Sho1, the upstream MAP kinase kinase kinase Ste11, and the downstream MAP kinase Hog1. Nonetheless, previous work has shown that Ste11 can be activated even when Pbs2 is deleted, resulting in inappropriate crosstalk to the mating pathway. We have found a region in the C terminus of Sho1 that binds Ste11 independently of Pbs2 and is required for crosstalk. These data support a model in which Sho1 has at least two separable interaction regions: one that binds Ste11 and mediates its activation, and one that binds Pbs2, directing Ste11 to act on Pbs2. Thus, a network of interactions provided by both Sho1 and Pbs2 appears to direct pathway information flow.

MAP Kinase Kinase Kinases↗

PubMed2004

Rewiring cell signaling: the logic and plasticity of eukaryotic protein circuitry.

Living cells rival computers in their ability to process external information and make complex behavioral decisions. Many of these decisions are made by networks of interacting signaling proteins. Ongoing structural, biochemical and cell-based studies have begun to reveal several common principles by which protein components are used to specifically transmit and process information. Recent engineering studies demonstrate that these relatively simple principles can be used to rewire signaling behavior in a process that mimics the evolution of new phenotypic responses.

Adaptation, Physiological↗

PubMed2003

Optimization of specificity in a cellular protein interaction network by negative selection.

Most proteins that participate in cellular signalling networks contain modular protein-interaction domains. Multiple versions of such domains are present within a given organism: the yeast proteome, for example, contains 27 different Src homology 3 (SH3) domains. This raises the potential problem of cross-reaction. It is generally thought that isolated domain-ligand pairs lack sufficient information to encode biologically unique interactions, and that specificity is instead encoded by the context in which the interaction pairs are presented. Here we show that an isolated peptide ligand from the yeast protein Pbs2 recognizes its biological partner, the SH3 domain from Sho1, with near-absolute specificity--no other SH3 domain present in the yeast genome cross-reacts with the Pbs2 peptide, in vivo or in vitro. Such high specificity, however, is not observed in a set of non-yeast SH3 domains, and Pbs2 motif variants that cross-react with other SH3 domains confer a fitness defect, indicating that the Pbs2 motif might have been optimized to minimize interaction with competing domains specifically found in yeast. System-wide negative selection is a subtle but powerful evolutionary mechanism to optimize specificity within an interaction network composed of overlapping recognition elements.

Amino Acid Motifs↗

PubMed2003

Reprogramming control of an allosteric signaling switch through modular recombination.

Many eukaryotic signaling proteins are composed of simple modular binding domains, yet they can display sophisticated behaviors such as allosteric gating and multi-input signal integration, properties essential for complex cellular circuits. To understand how such behavior can emerge from combinations of simple domains, we engineered variants of the actin regulatory protein N-WASP (neuronal Wiskott-Aldrich syndrome protein) in which the "output" domain of N-WASP was recombined with heterologous autoinhibitory "input" domains. Synthetic switch proteins were created with diverse gating behaviors in response to nonphysiological inputs. Thus, this type of modular framework can facilitate the evolution or engineering of cellular signaling circuits.

Actins↗

PubMed2003

The structure and function of proline recognition domains.

One particularly abundant group of modular recognition domains consists of those that bind proline-rich motifs. Such modules, including the SH3, WW, and EVH1 domains, play a critical role in the assembly and regulation of many intracellular signaling complexes. These domains use strikingly similar molecular mechanisms of proline recognition. We discuss some of the potential biological advantages conferred by proline recognition, which may explain its widespread use in signaling.

Animals↗

PubMed2003

Role of electrostatic interactions in PDZ domain ligand recognition.

PDZ domains are protein-protein interaction modules that normally recognize short C-terminal peptides. The apparent requirement for a ligand with a free terminal carboxylate group has led to the proposal that electrostatic interactions with the terminus play a significant role in recognition. However, this model has been called into question by the more recent finding that PDZ domains can recognize some internal peptide motifs that occur within a specific secondary structure context. Although these motifs bind at the same interface, they lack a terminal charge. Here we have investigated the role of electrostatics in PDZ-mediated recognition in the mouse alpha1-syntrophin PDZ domain by examining the salt dependence of binding to both terminal and internal ligands and the effects of mutating a conserved basic residue previously proposed to play a role in electrostatic recognition. These studies indicate that direct electrostatic interactions with the peptide terminus do not play a significant energetic role in binding. Additional chemical modification studies of the peptide terminus support a model in which steric and hydrogen bonding complementarity play a primary role in recognition specificity. Peptides with a free carboxy terminus, or presented within a specific structural context, can satisfy these requirements.

Amino Acid Sequence↗

PubMed2003

Rewiring MAP kinase pathways using alternative scaffold assembly mechanisms.

How scaffold proteins control information flow in signaling pathways is poorly understood: Do they simply tether components, or do they precisely orient and activate them? We found that the yeast mitogen-activated protein (MAP) kinase scaffold Ste5 is tolerant to major stereochemical perturbations; heterologous protein interactions could functionally replace native kinase recruitment interactions, indicating that simple tethering is largely sufficient for scaffold-mediated signaling. Moreover, by engineering a scaffold that tethers a unique kinase set, we could create a synthetic MAP kinase pathway with non-natural input-output properties. These findings demonstrate that scaffolds are highly flexible organizing factors that can facilitate pathway evolution and engineering.

Adaptor Proteins, Signal Transducing↗

PubMed2003

Complex inheritance of familial hypercholanemia with associated mutations in TJP2 and BAAT.

Familial hypercholanemia (FHC) is characterized by elevated serum bile acid concentrations, itching, and fat malabsorption. We show here that FHC in Amish individuals is associated with mutations in tight junction protein 2 (encoded by TJP2, also known as ZO-2) and bile acid Coenzyme A: amino acid N-acyltransferase (encoded by BAAT). The mutation of TJP2, which occurs in the first PDZ domain, reduces domain stability and ligand binding in vitro. We noted a morphological change in hepatic tight junctions. The mutation of BAAT, a bile acid-conjugating enzyme, abrogates enzyme activity; serum of individuals homozygous with respect to this mutation contains only unconjugated bile acids. Mutations in both TJP2 and BAAT may disrupt bile acid transport and circulation. Inheritance seems to be oligogenic, with genotype at BAAT modifying penetrance in individuals homozygous with respect to the mutation in TJP2.

Acyltransferases↗

PubMed2002

Structure of the N-WASP EVH1 domain-WIP complex: insight into the molecular basis of Wiskott-Aldrich Syndrome.

Missense mutants that cause the immune disorder Wiskott-Aldrich Syndrome (WAS) map primarily to the Enabled/VASP homology 1 (EVH1) domain of the actin regulatory protein WASP. This domain has been implicated in both peptide and phospholipid binding. We show here that the N-WASP EVH1 domain does not bind phosphatidyl inositol-(4,5)-bisphosphate, as previously reported, but does specifically bind a 25 residue motif from the WASP Interacting Protein (WIP). The NMR structure of the complex reveals a novel recognition mechanism-the WIP ligand, which is far longer than canonical EVH1 ligands, wraps around the domain, contacting a narrow but extended surface. This recognition mechanism provides a basis for understanding the effects of mutations that cause WAS.

Amino Acid Sequence↗

PubMed2002

Coordinated folding and association of the LIN-2, -7 (L27) domain. An obligate heterodimerization involved in assembly of signaling and cell polarity complexes.

LIN-2, -7 (L27) homology domains are putative protein-protein interaction modules found in several scaffold proteins involved in the assembly of polarized cell-signaling structures. These specific interaction pairs are well conserved across metazoan species, from worms to man. We have expressed and purified L27 domains from multiple species and find that certain domains from proteins such as Caenorhabditis elegans LIN-2 and LIN-7 can specifically heterodimerize. Biophysical analysis of interacting L27 domains demonstrates that the domains interact with a 1:1 stoichiometry. Circular dichroism studies reveal that the domains appear to function as an obligate heterodimer; individually the domains are largely unfolded, but when associated they show a significant increase in helicity, as well as a cooperative unfolding transition. These novel obligate interacting pairs are likely to play a key role in regulating the organization of signaling proteins at polarized cell structures.

Animals↗