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W A Lim

Publications and source records attributed to W A Lim.

At least 19 recordsLinked to original sources
PubMed2000

Integration of multiple signals through cooperative regulation of the N-WASP-Arp2/3 complex.

The protein N-WASP [a homolog to the Wiskott-Aldrich syndrome protein (WASP)] regulates actin polymerization by stimulating the actin-nucleating activity of the actin-related protein 2/3 (Arp2/3) complex. N-WASP is tightly regulated by multiple signals: Only costimulation by Cdc42 and phosphatidylinositol (4,5)-bisphosphate (PIP2) yields potent polymerization. We found that regulation requires N-WASP's constitutively active output domain (VCA) and two regulatory domains: a Cdc42-binding domain and a previously undescribed PIP(2)-binding domain. In the absence of stimuli, the regulatory modules together hold the VCA-Arp2/3 complex in an inactive "closed" conformation. In this state, both the Cdc42- and PIP2-binding sites are masked. Binding of either input destabilizes the closed state and enhances binding of the other input. This cooperative activation mechanism shows how combinations of simple binding domains can be used to integrate and amplify coincident signals.

Actin Cytoskeleton↗
PubMed2000

Improving SH3 domain ligand selectivity using a non-natural scaffold.

BACKGROUND: Src homology 3 (SH3) domains bind sequences bearing the consensus motif PxxP (where P is proline and x is any amino acid), wherein domain specificity is mediated largely by sequences flanking the PxxP core. This specificity is limited, however, as most SH3 domains show high ligand cross-reactivity. We have recently shown that diverse N-substituted residues (peptoids) can replace the prolines in the PxxP motif, yielding a new source of ligand specificity. RESULTS: We have tested the effects of combining multiple peptoid substitutions with specific flanking sequences on ligand affinity and specificity. We show that by varying these different elements, a ligand can be selectively tuned to target a single SH3 domain in a test set. In addition, we show that by making multiple peptoid substitutions, high-affinity ligands can be generated that completely lack the canonical PxxP motif. The resulting ligands can potently disrupt natural SH3-mediated interactions. CONCLUSIONS: Peptide-peptoid hybrid scaffolds yield SH3 ligands with markedly improved domain selectivity, overcoming one of the principal challenges in designing inhibitors against these domains. These compounds represent important leads in the search for orthogonal inhibitors of SH3 domains, and can serve as tools for the dissection of complex signaling pathways.

Amino Acid Motifs↗
PubMed1999

PSD-95 assembles a ternary complex with the N-methyl-D-aspartic acid receptor and a bivalent neuronal NO synthase PDZ domain.

Nitric oxide (NO) biosynthesis in cerebellum is preferentially activated by calcium influx through N-methyl-D-aspartate (NMDA)-type glutamate receptors, suggesting that there is a specific link between these receptors and neuronal NO synthase (nNOS). Here, we find that PSD-95 assembles a postsynaptic protein complex containing nNOS and NMDA receptors. Formation of this complex is mediated by the PDZ domains of PSD-95, which bind to the COOH termini of specific NMDA receptor subunits. In contrast, nNOS is recruited to this complex by a novel PDZ-PDZ interaction in which PSD-95 recognizes an internal motif adjacent to the consensus nNOS PDZ domain. This internal motif is a structured "pseudo-peptide" extension of the nNOS PDZ that interacts with the peptide-binding pocket of PSD-95 PDZ2. This asymmetric interaction leaves the peptide-binding pocket of the nNOS PDZ domain available to interact with additional COOH-terminal PDZ ligands. Accordingly, we find that the nNOS PDZ domain can bind PSD-95 PDZ2 and a COOH-terminal peptide simultaneously. This bivalent nature of the nNOS PDZ domain further expands the scope for assembly of protein networks by PDZ domains.

Animals↗
PubMed1999

Structure of the enabled/VASP homology 1 domain-peptide complex: a key component in the spatial control of actin assembly.

The Enabled/VASP homology 1 (EVH1; also called WH1) domain is an interaction module found in several proteins implicated in actin-based cell motility. EVH1 domains bind the consensus proline-rich motif FPPPP and are required for targeting the actin assembly machinery to sites of cytoskeletal remodeling. The crystal structure of the mammalian Enabled (Mena) EVH1 domain complexed with a peptide ligand reveals a mechanism of recognition distinct from that used by other proline-binding modules. The EVH1 domain fold is unexpectedly similar to that of the pleckstrin homology domain, a membrane localization module. This finding demonstrates the functional plasticity of the pleckstrin homology fold as a binding scaffold and suggests that membrane association may play an auxiliary role in EVH1 targeting.

Actins↗
PubMed1999

Unexpected modes of PDZ domain scaffolding revealed by structure of nNOS-syntrophin complex.

The PDZ protein interaction domain of neuronal nitric oxide synthase (nNOS) can heterodimerize with the PDZ domains of postsynaptic density protein 95 and syntrophin through interactions that are not mediated by recognition of a typical carboxyl-terminal motif. The nNOS-syntrophin PDZ complex structure revealed that the domains interact in an unusual linear head-to-tail arrangement. The nNOS PDZ domain has two opposite interaction surfaces-one face has the canonical peptide binding groove, whereas the other has a beta-hairpin "finger." This nNOS beta finger docks in the syntrophin peptide binding groove, mimicking a peptide ligand, except that a sharp beta turn replaces the normally required carboxyl terminus. This structure explains how PDZ domains can participate in diverse interaction modes to assemble protein networks.

Amino Acid Sequence↗
PubMed1998

Exploiting the basis of proline recognition by SH3 and WW domains: design of N-substituted inhibitors.

Src homology 3 (SH3) and WW protein interaction domains bind specific proline-rich sequences. However, instead of recognizing critical prolines on the basis of side chain shape or rigidity, these domains broadly accepted amide N-substituted residues. Proline is apparently specifically selected in vivo, despite low complementarity, because it is the only endogenous N-substituted amino acid. This discriminatory mechanism explains how these domains achieve specific but low-affinity recognition, a property that is necessary for transient signaling interactions. The mechanism can be exploited: screening a series of ligands in which key prolines were replaced by nonnatural N-substituted residues yielded a ligand that selectively bound the Grb2 SH3 domain with 100 times greater affinity.

Adaptor Proteins, Signal Transducing↗
PubMed1994

Structural determinants of peptide-binding orientation and of sequence specificity in SH3 domains.

The Src-homology-3 (SH3) domains of the Caenorhabditis elegans protein SEM-5 and its human and Drosophila homologues, Grb2 and Drk (refs 1-4), bind proline-rich sequences found in the nucleotide-exchange factor Sos as part of their proposed function linking receptor tyrosine kinase activation to Ras activation. Here we report the crystal structure at 2.0 A resolution of the carboxy-terminal SH3 domain from SEM-5 complexed to the mSos-derived amino-acid sequence PPPVPPRRR. The peptide is found to bind in an orientation ('minus') that is precisely opposite to that observed previously ('plus' orientation) in other SH3-peptide complexes. This novel ability of peptide-recognition proteins to recognize peptides in two distinct modes may play an important role in the signalling specificity of pathways involving SH3 domains. Comparison of this structure with other SH3 complexes reveals how a conserved binding face can be used to recognize peptides in different orientations, and why the Sos peptide binds in this particular orientation.

Amino Acid Sequence↗
PubMed1994

The crystal structure of a mutant protein with altered but improved hydrophobic core packing.

The dense packing observed in protein interiors appears to be crucial for stabilizing the native structure--even subtle internal substitutions are usually destabilizing. Thus, steric complementarity of core residues is thought to be an important criterion for "inverse folding" predictive methods, which judge whether a newly determined sequence is consistent with any known folds. A major problem in the development of useful core packing evaluation algorithms, however, is that there are occasional mutations that are predicted to disrupt native packing but that yield an equally or more stable protein. We have solved the crystal structure of such a variant of lambda repressor, which, despite having three larger core substitutions, is more stable than the wild type. The structure reveals that the protein accommodates the potentially disruptive residues with shifts in its alpha-helical arrangement. The variant is apparently more stable because its packing is improved--the core has a higher packing density and little geometric strain. These rearrangements, however, cause repositioning of functional residues, which result in reduced DNA binding activity. By comparing these results with the predictions of two core packing algorithms, it is clear that the protein possesses a relatively high degree of main-chain flexibility that must be accounted for in order to predict the full spectrum of compatible core sequences. This study also shows how, in protein evolution, a particular set of core residue identities might be selected not because they provide optimal stability but because they provide sufficient stability in addition to the precise structure required for optimal activity.

Bacteriophage lambda↗
PubMed1994

Stability and peptide binding affinity of an SH3 domain from the Caenorhabditis elegans signaling protein Sem-5.

We have determined the thermodynamic stability and peptide binding affinity of the carboxy-terminal Src homology 3 (SH3) domain from the Caenorhabditis elegans signal-transduction protein Sem-5. Despite its small size (62 residues) and lack of disulfide bonds, this domain is highly stable to thermal denaturation--at pH 7.3, the protein has a Tm of 73.1 degrees C. Interestingly, the protein is not maximally stable at neutral pH, but reaches a maximum at around pH 4.7 (Tm approximately equal to 80 degrees C). Increasing ionic strength also stabilizes the protein, suggesting that 1 or more carboxylate ions are involved in a destabilizing electrostatic interaction. By guanidine hydrochloride denaturation, the protein is calculated to have a free energy of unfolding of 4.1 kcal/mol at 25 degrees C. We have also characterized binding of the domain to 2 different length proline-rich peptides from the guanine nucleotide exchange factor, Sos, one of Sem-5's likely physiological ligands in cytoplasmic signal transduction. Upon binding, these peptides cause about a 2-fold increase in fluorescence intensity. Both bind with only modest affinities (Kd approximately equal to 30 microM), lower than some previous estimates for SH3 domains. By fluorescence, the domain also appears to associate with the homopolymer poly-L-proline in a similar fashion.

Amino Acid Sequence↗
PubMed1994

Critical residues in an SH3 domain from Sem-5 suggest a mechanism for proline-rich peptide recognition.

Src homology 3 (SH3) domains bind specific proline-rich peptide motifs. To identify interactions involved in peptide recognition, we have mutated residues on the putative binding surface of an SH3 domain from the Caenorhabditis elegans protein Sem-5. Among the most critical positions are three adjacent aromatic residues, which appear to participate in highly stereospecific packing interactions with the ligand. The co-planar arrangement of two of these residues closely matches the periodicity of a poly-proline II (PPII) helix. Thus, a model for recognition has the peptide adopting a PPII helix, with the pyrrolidine rings on one helical face interlocking with the aromatic SH3 residues.

Amino Acid Sequence↗
PubMed1992

Structural and energetic consequences of disruptive mutations in a protein core.

We have characterized the properties of a set of variants of the N-terminal domain of lambda repressor bearing disruptive mutations in the hydrophobic core. These mutations include some that dramatically alter the total core residue volume (by up to six methylene groups) and some that place a single polar residue into the otherwise hydrophobic core. The structural properties of the purified proteins have been studied by CD spectroscopy, biological activity, recognition by conformation-specific monoclonal antibodies, and 1H NMR spectroscopy. The stabilities of the proteins have been measured by thermal and guanidine hydrochloride denaturation. Proteins with disruptive core mutations are found to display a continuum of increasingly nonnative properties. Large internal volume changes cause both significant conformational rearrangements and destabilization by up to 5 kcal/mol. Variants with polar substitutions at core positions no longer behave like well-folded proteins but rather display characteristics of molten globules. However, even proteins bearing some of the most disruptive mutations retain many of the crude secondary and tertiary structural features of the wild-type protein. These results indicate that primitive elements of native structure can form in the absence of normal core packing.

Amino Acid Sequence↗
PubMed1991

The role of internal packing interactions in determining the structure and stability of a protein.

Cassette mutagenesis has been used to investigate how internal packing interactions help to specify a protein's three-dimensional structure and stability. Three interacting residues in the hydrophobic core of the N-terminal domain of lambda repressor were randomized combinatorially. The randomization was restricted to the five amino acids Val, Leu, Ile, Met and Phe, thereby generating a sterically diverse set of core sequences composed solely of hydrophobic residues. We have isolated 78 of the 125 possible sequences generated by this randomization. Approximately 70% of the isolated sequences show some level of biological activity, and thus still carry sufficient information to encode the basic structure of lambda repressor. An assay based on the temperature dependence of activity in vivo has been used to estimate the relative activities and thermal stabilities of the set of mutants. In addition, nine mutants have been purified and their stabilities and DNA binding activities characterized in vitro. Of the 56 active sequences, only two, in addition to the wild-type, maintain the wild-type level of stability and activity. All three of these proteins satisfy stringent requirements for specifically shaped residues at each position. All of the remaining active sequences have reduced stabilities and/or reduced DNA binding affinities. These and previous results suggest that there are two levels of structural information encoded in core residues. At the first level, the basic structural information appears to reside largely in the hydrophobic character of these residues. The majority of sequences that simply maintain hydrophobicity at core positions are able to adopt the overall lambda repressor fold and maintain moderate stability. At the second, more detailed level, specific steric features of these residues and their packing interactions clearly act as important determinants of the protein's precise structure and stability. These results imply that many of the basic structural features of a protein could be predicted from relatively simple, degenerate sequence patterns.

DNA, Bacterial↗