Mickey Kosloff

Sequence-similar, structure-dissimilar protein pairs in the PDB.

It is often assumed that in the Protein Data Bank (PDB), two proteins with similar sequences will also have similar structures. Accordingly, it has proved useful to develop subsets of the PDB from which “redundant” structures have been removed, based on a sequence‐based criterion for similarity. Similarly, when predicting protein structure using homology modeling, if a template structure for modeling a target sequence is selected by sequence alone, this implicitly assumes that all sequence‐similar templates are equivalent. Here, we show that this assumption is often not correct and that standard approaches to create subsets of the PDB can lead to the loss of structurally and functionally important information. We have carried out sequence‐based structural superpositions and geometry‐based structural alignments of a large number of protein pairs to determine the extent to which sequence similarity ensures structural similarity. We find many examples where two proteins that are similar in sequence have structures that differ significantly from one another. The source of the structural differences usually has a functional basis. The number of such proteins pairs that are identified and the magnitude of the dissimilarity depend on the approach that is used to calculate the differences; in particular sequence‐based structure superpositioning will identify a larger number of structurally dissimilar pairs than geometry‐based structural alignments. When two sequences can be aligned in a statistically meaningful way, sequence‐based structural superpositioning provides a meaningful measure of structural differences. This approach and geometry‐based structure alignments reveal somewhat different information and one or the other might be preferable in a given application. Our results suggest that in some cases, notably homology modeling, the common use of nonredundant datasets, culled from the PDB based on sequence, may mask important structural and functional information. We have established a data base of sequence‐similar, structurally dissimilar protein pairs that will help address this problem (http://luna.bioc.columbia.edu/rachel/seqsimstrdiff.htm). Proteins 2008.

Substrate assisted catalysis – application to G proteins

The idea that both the substrate and the enzyme contribute to catalysis (substrate assisted catalysis; SAC) is applicable to guanine nucleotide-binding proteins (G proteins). Naturally occurring SAC uses GTP as a general base in the GTPase reaction catalyzed by G proteins. Engineered SAC has identified a putative rate-limiting step for the GTPase reaction and shown that GTPase-deficient oncogenic Ras mutants are not irreversibly impaired. Thus, anti-cancer drugs could potentially be designed to restore the blocked GTPase reaction.

Electrostatic and lipid-anchor contributions to the interaction of transducin with membranes: Mechanistic implications for activation and translocation

The heterotrimeric G protein transducin is a key component of the vertebrate phototransduction cascade. Transducin is peripherally attached to membranes of the rod outer segment, where it interacts with other proteins at the membrane-cytosol interface. However, upon sustained activation by light, the dissociated Gtα and Gβ1γ1 subunits of transducin translocate from the outer segment to other parts of the rod cell. Here we used a computational approach to analyze the interaction strength of transducin and its subunits with acidic lipid bilayers, as well as the range of orientations that they are allowed to occupy on the membrane surface. Our results suggest that the combined constraints of electrostatics and lipid anchors substantially limit the rotational degrees of freedom of the membrane-bound transducin heterotrimer. This may contribute to a faster transducin activation rate by accelerating transducin-rhodopsin complex formation. Notably, the membrane interactions of the dissociated transducin subunits are very different from those of the heterotrimer. As shown previously, Gβ1γ1 experiences significant attractive interactions with negatively charged membranes, whereas our new results suggest that Gtα is electrostatically repelled by such membranes. We suggest that this repulsion could facilitate the membrane dissociation and intracellular translocation of Gtα. Moreover, based on similarities in sequence and electrostatic properties, we propose that the properties described for transducin are common to its homologs within the Gi subfamily. In a broader view, this work exemplifies how the activity-dependent association and dissociation of a G protein can change both the affinity for membranes and the range of allowed orientations, thereby modulating G protein function.

Regulator of G-protein signaling-21 (RGS21) is an inhibitor of bitter gustatory signaling found in lingual and airway epithelia.

Background: RGS21 is expressed in tastant-responsive lingual epithelium, but with unknown function. Results: RGS21 accelerated intrinsic GTPase activity of multiple Gα subunits; RGS21 over- and underexpression in epithelial cells modulated bitterant responsiveness. Conclusion: RGS21 is a negative regulator of bitterant signal transduction. Significance: RGS21 represents a nonreceptor regulatory component of gustatory signaling that alters sensitivity of bitterant responsiveness in an endogenous, cellular context. The gustatory system detects tastants and transmits signals to the brain regarding ingested substances and nutrients. Although tastant receptors and taste signaling pathways have been identified, little is known about their regulation. Because bitter, sweet, and umami taste receptors are G protein-coupled receptors (GPCRs), we hypothesized that regulators of G protein signaling (RGS) proteins may be involved. The recent cloning of RGS21 from taste bud cells has implicated this protein in the regulation of taste signaling; however, the exact role of RGS21 has not been precisely defined. Here, we sought to determine the role of RGS21 in tastant responsiveness. Biochemical analyses confirmed in silico predictions that RGS21 acts as a GTPase-accelerating protein (GAP) for multiple G protein α subunits, including adenylyl cyclase-inhibitory (Gαi) subunits and those thought to be involved in tastant signal transduction. Using a combination of in situ hybridization, RT-PCR, immunohistochemistry, and immunofluorescence, we demonstrate that RGS21 is not only endogenously expressed in mouse taste buds but also in lung airway epithelial cells, which have previously been shown to express components of the taste signaling cascade. Furthermore, as shown by reverse transcription-PCR, the immortalized human airway cell line 16HBE was found to express transcripts for tastant receptors, RGS21, and downstream taste signaling components. Over- and underexpression of RGS21 in 16HBE cells confirmed that RGS21 acts to oppose bitter tastant signaling to cAMP and calcium second messenger changes. Our data collectively suggests that RGS21 modulates bitter taste signal transduction.

Cooperative phosphoinositide and peptide binding by PSD-95/discs large/ZO-1 (PDZ) domain of polychaetoid, Drosophila zonulin

Background: PDZ domains mediate protein interaction but may also sense lipid signaling. Results: We characterized a new phosphatidylinositol 4,5-bisphosphate-interacting PDZ domain by in vitro, in vivo, and in silico approaches. Conclusion: Membrane binding and subcellular localization of this domain is achieved by a combination of peptide and lipid interactions. Significance: PDZ domains might support cooperative detection of peptide and lipid in cell signaling. PDZ domains are well known protein-protein interaction modules that, as part of multidomain proteins, assemble molecular complexes. Some PDZ domains have been reported to interact with membrane lipids, in particular phosphatidylinositol phosphates, but few studies have been aimed at elucidating the prevalence or the molecular details of such interactions. We screened 46 Drosophila PDZ domains for phosphoinositide-dependent cellular localization and discovered that the second PDZ domain of polychaetoid (Pyd PDZ2) interacts with phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) at the plasma membrane. Surface plasmon resonance binding experiments with recombinant protein established that Pyd PDZ2 interacts with phosphatidylinositol phosphates with apparent affinities in the micromolar range. Electrostatic interactions involving an extended positively charged surface of Pyd PDZ2 are crucial for the PtdIns(4,5)P2-dependent membrane interactions as shown by a combination of three-dimensional modeling, mutagenesis, binding, and localization studies. In vivo localization studies further suggested that both lipid and peptide binding contribute to membrane localization. We identified the transmembrane protein Crumbs as a Pyd PDZ2 ligand and probed the relation between peptide and PtdIns(4,5)P2 binding. Contrary to the prevalent view on PDZ/peptide/lipid binding, we did not find competition between peptide and lipid ligands. Instead, preloading the protein with the 10-mer Crb3 peptide increased the apparent affinity of Pyd PDZ2 for PtdIns(4,5)P2 6-fold. Our results suggest that membrane localization of Pyd PDZ2 may be driven by a combination of peptide and PtdIns(4,5)P2 binding, which raises the intriguing possibility that the domain may coordinate protein- and phospholipid-mediated signals.

PLoS Computational Biology conference postcards from ISMB 2010.

The annual international conference on Intelligent Systems for Molecular Biology (ISMB) is the largest meeting of the International Society for Computational Biology (ISCB). In 2010 it was held in Boston, United States, July 11–13. What follows are four conference postcards that reflect different activities considered exciting and important by younger attendees. Postcards, as the name suggests, are brief reports on the talks and other events that interested attendees. You can read more about the idea of conference postcards at http://www.ploscompbiol.org/doi/pcbi.1000746, and if you are a graduate student or postdoctoral fellow, please consider contributing postcards at any future meetings of interest to the PLoS Computational Biology readership. We want to hear your view of the science being presented.

Substrate‐assisted catalysis: Implications for biotechnology and drug design

In enzyme catalysis, the convention is that the enzyme supplies all the functional groups that are needed to convert a substrate into a product. This convention, however, is now recognized to have some exceptions. In a growing number of cases it is evident that the substrate also provides one or more functional groups that actively participate in the catalytic process. These cases are grouped together under the title “substrate‐assisted catalysis” (SAC). Examples of SAC have been described both for native and for engineered enzymes that were rendered inactive by mutations. Such mutations eliminate amino acid sidechains that participate in the catalytic process and thereby cause partial or total loss of enzymatic activity. For several of these mutant enzymes, a modified substrate bearing functional groups similar to those that had been eliminated by the mutation was found to rescue enzyme activity. A notable example is the mutual specificity found between mutant serine proteases and their modified substrates. This creates a highly specific site for proteolytic cleavage, a desirable property in the processing of recombinant fusion products. An attractive target for SAC is the G‐protein family. It was applied to two of its members—Gsα and p21‐Ras. In both cases it was possible to restore the GTPase activity of the mutants back to the level of the wild‐type proteins. Beyond restoring activity by SAC, further modifications of the substrate were introduced to support or refute particular roles of the functional groups in the GTPase reaction. This approach was also applied as a molecular tool to discriminate between specific enzymatic mechanisms and as a guideline to incorporate particular functional groups into the substrate. Taken together, these studies pave the way to novel therapeutic and biotechnological approaches aimed at restoring the activity of mutant inactive enzymes. Drug Dev. Res. 50:250–257, 2000.