Papers (circa 2013)

Protein structure function computational tools  

From Protein Structure to Function via Computational Tools and Approaches.

Rachel Kolodny and Mickey Kosloff
Isr. J. Chem. (2013) In Press.
web pdf (1.5Mb)

The 3D structures of proteins are often considered fundamental for understanding their function. Yet, because of the complexity of protein structure, extracting specific functional information from structures can be a considerable challenge. Here, we present selected approaches and tools that were developed in the Kolodny and Kosloff labs to study and connect protein sequence, structure, and function spaces.

First, we consider a global perspective of structure space and view the protein data bank (PDB) as a database. We highlight challenges in searching protein structure space and in using the PDB as the starting point for computational structural studies. Then we describe a function-oriented view and show examples of how multiple protein structures can be used to extract insights about the function and specificity of proteins at the family level.

RGS12 gastducin localization  

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

Staci Cohen, Brian Buckley, Mickey Kosloff, Alaina Garland, Dustin Bosch, Gang Cheng Jr, Harish Radhakrishna, Michael Brown, Francis Willard, Vadim Arshavsky, Robert Tarran, David Siderovski and Adam Kimple
J. Biol. Chem. (2012) 287 (50): 41706-41719.
web pdf (4.7Mb)

RGS21 is a newly discovered RGS protein, whose biological function and specificity are unclear. The recent cloning of RGS21 from taste bud cells has implicated this protein in the regulation of taste signaling presumably via the Gi family members gustducin and transducin (Gt), which have been implicated in these sensory cascades. However, the exact role of RGS21 has not been precisely defined.

Here, we sought to determine the role of RGS21 in tastant responsiveness. Our computational analysis predicted that RGS21 is a highly active and promiscuous GAP toward members of the Gi subfamily. Indeed, our biochemical experiments confirmed that RGS21 shows high GAP activity towards Gt and Go. We further demonstrated 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, 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.


Compartmentalization and Ca2+ buffering are essential for prevention of light-induced retinal degeneration.

Shirley Weiss, Elkana Kohn, Daniela Dadon, Ben Katz, Maximilian Peters, Mario Lebendiker, Mickey Kosloff, Nansi Jo Colley and Baruch Minke
J. Neurosci. (2012) 32 (42): 14696-14708.
web pdf (3.6Mb)

Fly photoreceptors are polarized cells, each of which has an extended interface between its cell body and the light-signaling compartment, the rhabdomere. Upon intense illumination, rhabdomeric calcium concentration reaches millimolar levels that would be toxic if calcium diffusion between the rhabdomere and cell body was not robustly attenuated. Yet, it is not clear how such effective attenuation is obtained.

Here, we show that calcium homeostasis in the photoreceptor cell relies on the protein calphotin, which has a unique amino acid composition and is predicted to be intrinsically-unstructured. This unusual protein functions as an immobile calcium buffer localized along the base of the rhabdomere, separating the signaling compartment from the cell body. Generation and analyses of transgenic Drosophila strains, in which calphotin-expression levels were reduced in a graded manner, showed that moderately reduced calphotin expression impaired calcium homeostasis while calphotin elimination resulted in severe light-dependent photoreceptor degeneration. The degeneration was rescued by prevention of calcium overload via overexpression of CalX, the Na(+)-Ca(2+) exchanger. In addition, reduced calphotin levels resulted in abnormally fast kinetics of calcium elevation in photoreceptor cells. Together, our data suggest that calphotin functions as a calcium buffer. We propose that calphotin-mediated compartmentalization and calcium buffering constitute an effective strategy to protect cells from calcium overload and light-induced degeneration. Further analysis of Calphotin's sequence and amino acid composition raises the intriguing possibility that calphotin is a prototypic example of a new and diverse family of intrinsically-unstructured calcium-binding proteins in diverse organisms.

Pyd-PDZ2-membrane interactions  

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

Ylva Ivarsson, Anna Maria Wawrzyniak, Gunther Wuytens, Mickey Kosloff, Elke Vermeiren, Marie Raport and Pascale Zimmermann
J. Biol. Chem. (2011) 286 (52): 44669-44678.
web pdf (3.3Mb)

PDZ domains are well known protein-protein interaction modules that assemble molecular complexes as part of multidomain proteins. Some PDZ domains have been reported to interact with membrane lipids (particularly phosphoinositides), but few studies have elucidated the molecular details of such interactions.

Here, 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 at the plasma membrane. Surface plasmon resonance binding experiments established that Pyd PDZ2 interacts with phosphoinositides with micromolar range affinity. We then analyzed a homology model of dimeric Pyd PDZ2 and showed that electrostatic interactions involving an extended positively charged surface of Pyd PDZ2 are crucial for its phosphoinositide-dependent membrane interactions. We then validated these predictions by mutagenesis studies followed by binding and localization experiments. Furthermore, we show that both lipid and peptide binding contribute to the membrane localization of Pyd PDZ2, raising the intriguing possibility that this PDZ domain may coordinate protein- and phospholipid-mediated signals.

RGS/G-protein specificity  

Integrating energy calculations with functional assays to decipher the specificity of G protein-RGS protein interactions.

Mickey Kosloff, Amanda Travis, Dustin Bosch, David Siderovski and Vadim Arshavsky
Nat. Struct. Mol. Biol. (2011) 18 (7): 846-853.
web pdf (2.3Mb) Sup. Materials (600Kb)

External coverage:
F1000 evaluation HFSP coverage

We describe an approach to address a fundamental challenge in the field of signal transduction - deciphering how protein structure encodes specific interactions between large protein families. As a model system to study specificity, we chose to investigate the interactions of heterotrimeric G-proteins with Regulators of G-protein Signaling (RGSs). RGSs are responsible for turning G-proteins "off" and in multiple cascades across most human tissues RGSs determine the duration of the G-protein coupled signaling. RGSs have also been implicated in a wide range of human pathologies and are promising drug targets, both as primary targets and as complements to drugs that target other components in G-protein signaling (such as G-protein Coupled Receptors).

To understand how RGS protein structure encodes both their common ability to inactivate G-proteins but also their selective G-protein recognition, we integrated structure-based energy calculations with biochemical measurements of RGS protein activity. Using a consensus approach across the eight available RGS-domain/G-protein crystal structures, we established a structure-to-sequence map predicting which RGS residues are essential for function and which RGS residues can modulate specific interactions with the cognate G-protein. This map revealed that, in addition to previously identified conserved residues, RGS proteins contain another group of variable "Modulatory Residues", which reside at the periphery of the RGS-domain/G-protein interface and fine-tune G-protein recognition. Mutations of Modulatory Residues in high-activity RGS proteins impaired RGS function, whereas redesign of low-activity RGS proteins in critical Modulatory positions yielded complete gain-of-function RGS mutants. Therefore, RGS proteins combine a conserved core interface with peripheral Modulatory Residues to selectively optimize G-protein recognition and inactivation.

Finally, we applied this computational approach to a completely different system - the interactions of colicin E7 with its inhibitory immunity proteins, a well-established model for studying protein-protein interaction specificity. We thereby showed the generality of our structure-based method and its potential use in a scalable "bottom-up" approach to study the structural basis for the "wiring" of signal transduction networks.

Interactions of transducin with membranes  

Electrostatic and Lipid-Anchor Contributions to the Interaction of Transducin with Membranes: Mechanistic Implications for Activation and Translocation.

Mickey Kosloff, Emil Alexov, Vadim Arshavsky and Barry Honig
J. Biol. Chem. (2008) 283 (45): 31197-31207.
web pdf (900 Kb) Sup. Materials (1.3 Mb)

The heterotrimeric G-protein transducin is a key component of the vertebrate phototransduction cascade. Like many other signal transduction proteins, transducin is peripherally attached to membranes of the rod outer segment, where it interacts with other proteins at the membrane-cytosol interface.

Here, we used a computational approach to analyze the interaction strength of transducin and its subunits with membranes, as well as the range of orientations that they are allowed to occupy on the membrane surface. We show that the membrane-bound transducin heterotrimer is constrained to a limited range of orientations, which can accelerate transducin's activation by rhodopsin. Notably, the membrane interactions of the dissociated transducin subunits are very different from those of the heterotrimer. While the beta-gamma complex is attracted to the negatively charged membrane, we show that Gt-alpha is electrostatically repelled by such membranes. We suggest that this repulsion could facilitate the membrane dissociation and intracellular translocation of Gt-alpha. Moreover, we show 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. Importantly, our approach can characterize quantitatively the interactions of other peripheral membrane proteins with membranes.

Sequence-similar, structure-dissimilar proteins in the PDB  

Sequence-Similar, Structure-Dissimilar Proteins in the PDB.

Mickey Kosloff and Rachel Kolodny
Proteins: Structure, function and Bioinformatics. (2008) 71 (2): 891-902.
web pdf (500 Kb) Sup. Materials (250 kb)

It is often assumed that in the Protein Data Bank (PDB), two proteins with similar sequences will also have similar structures. This assumption underlies many computational studies and structure prediction methods.

Here, we compare sequence-based structural superpositions and geometry-based structural alignments and show that the former provides a better measure of structure dissimilarity. Using sequence-based structural superpositioning we find many examples in the PDB where two proteins that are similar in sequence have structures that differ significantly from one another, usually in direct relation to their function. We conclude that the assumption of two proteins with similar sequences having similar structures is often incorrect and can lead to the loss of structurally and functionally important information.

We have established a database of sequence-similar, structurally dissimilar protein pairs that will help address this problem and show how this database can assist in predicting structures using homology modeling.

GST comparative structural comparison  

Comparative Structural Analysis of a Novel Glutathoine S-transferase (Atu5508) from Agrobacterium tumefaciens at 2.0 Angstrom Resolution.

Mickey Kosloff et al. (with JCSG consortium)
Proteins: Structure, function and Bioinformatics. (2006) 65 (3): 527-537.
web pdf (1 Mb)

Glutathione S-transferases (GSTs) comprise a diverse superfamily of enzymes found in organisms from all kingdoms of life. They are involved in diverse processes, notably small-molecule biosynthesis and detoxification, and are frequently used in protein engineering studies and as biotechnology tools. Because the GST superfamily is very diverse, GSTs have been subdivided into an ever-increasing number of sub-families, or “classes”, associated with different functionalities and enzymatic specificities. This classification has usually been based on a combination of criteria, such as biochemical properties, primary, tertiary and quaternary structure and immunological reactivity.

Here, through use of comparative sequence and structural analysis of the GST superfamily, we identified local sequence and structural signatures that allowed us to distinguish between different GST classes. Uniquely, this approach enables classifying novel GST proteins based on structure only, without requiring additional biochemical or immunological data.

In this work we also report the high-resolution X-ray structure of Atu5508, a putative GST from the pathogenic soil bacterium Agrobacterium tumefaciens (atGST1, PDB id 2FNO). Our comparative structural analysis suggests that atGST1 defines a new GST class, distinct from previously characterized GSTs both in structure and in function, which makes it an attractive target for further biochemical studies.

Importantly, our comparative analysis and characterization of atGST1 were first performed without any knowledge of the experimental structure, when we were “blindly“ predicting atGST1's 3D structure during CASP6. At the time, all available GST structures (templates) had less than 20% sequence identity to atGST1, yet we were able to successfully predict both the 3D structure and its functionality. Later, our approach and conclusions were corroborated using the experimental structure, thus validating the use of this approach to predict active site specificity.

G-protein active site comparison  

GTPase Catalysis by Ras and Other G-proteins: Insights from Substrate Directed SuperImposition.

Mickey Kosloff and Zvi Selinger
J. Mol. Biol. (2003) 331 (5): 1157-1170.
web pdf (2.5 Mb)

Comparisons of different protein structures are commonly carried out by superimposing the coordinates of the protein backbones or selected parts of the proteins. However, when the objective is analysis of similarities and differences in enzyme active sites, there is an inherent problem in using the domains under investigation for the superimposition. In this work we use a comparative approach we termed “Substrate Directed SuperImposition” (SDSI). It entails the superimposition of multiple protein-substrate structures using exclusively the coordinates of the comparable substrates. SDSI has the advantage of unbiased comparison of the active-site environment from the substrate’s point of view.

Here we apply SDSI to various G-protein structures for dissecting the mechanism of the GTPase reaction. SDSI indicates that dissimilar G-proteins stabilize the transition state of the GTPase reaction similarly and supports the commonality of the critical step in GTPase - the reorientation of the critical arginine and glutamine. We ascribe the catalytic inefficiency of the small G-protein Ras to the great flexibility of its active site and downplay possible catalytic roles for the Lys16 residue in GTPase catalysis. We also show that in contrast to all other Gly12 Ras mutants, which are oncogenic, the Gly12->Pro mutant does not interfere with the catalytic orientation of the critical glutamine. This suggests why this mutant has a higher rate of GTP hydrolysis and is non-transforming. Finally, we use SDSI to compare enzymes with very different 3D structures to reveal surprising similarities in the divergent catalytic machineries of G-proteins and UMP/CMP kinases.

Gq translocation in Drosophila photoreceptors  

Regulation of Light-dependent Gq-alpha Translocation and Morphological Changes in Fly Photoreceptors

Mickey Kosloff, Natalie Elia, Tamar Joel-Almagor, Rina Timberg, Troy Zars, David Hyde, Baruch Minke and Zvi Selinger
EMBO J. (2003) 22 (3): 459-468.
web1 web2 pdf (400 Kb)

Heterotrimeric G-proteins relay signals between membrane-bound G-protein coupled receptors (GPCRs) and downstream effectors. To perform their signaling function, G-proteins require anchorage to the plasma membrane. While in vitro and cell line based investigations suggested that the membrane localization of G-proteins is reversible, the results are contradictory. Importantly, these phenomena have not been characterized in vivo and the regulation of G-alpha localization within the natural endogenous environment of a specialized signaling cell is therefore of great interest.

Here we show, using live Drosophila flies, that light causes massive and reversible translocation of the visual Gq-alpha subunit from the membrane to the cytosol. This translocation is associated with marked architectural changes in the signaling compartment. We characterize the translocation cycle and how signaling molecules that interact with Gq-alpha regulate these processes. We also show that Gq-alpha is necessary but not sufficient to bring about the morphological changes in the signaling organelle. Furthermore, mutant analysis indicates that Gq-beta is essential for targeting of Gq-alpha to the membrane and suggests that Gq-beta is also needed for efficient activation of Gq-alpha by rhodopsin.

basic electrostatic motif in heterotrimeric G-proteins  

Structural Homology Discloses a Bifunctional Structural Motif at the N-termini of G alpha Proteins.

Mickey Kosloff, Natalie Elia and Zvi Selinger
Biochemistry (2002) 41 (49): 14518-14523.
web pdf (800 Kb)

Lipid modification by palmitoylation is a fundamental contributor to the membrane localization of heterotrimeric G-proteins and other proteins, but the signals leading to this reversible modification are still unknown.

Here, we use homology models of different human G-alpha paralogs (generated with automated methods) to identify a basic, positively charged structural motif in the N-termini of these proteins. We also show that a similar structural motif is found in other palmitoylated proteins. This basic motif is not readily discernible from sequence alone and is found in all palmitoylated-only G-proteins. Contrastingly, G-alpha subunits that also undergo myristoylation do not contain these prominent basic patches, suggesting that this basic motif and myristoylation play overlapping roles in membrane targeting.

Substrate assisted catalysis in G-proteins  

Substrate Assisted Catalysis - Application to G-proteins.

Mickey Kosloff and Zvi Selinger
TiBS (2001) 26 (3): 161-168.
web pdf (720 Kb)

In Substrate-Assisted Catalysis (SAC) the substrate for enzymatic catalysis provides one or more functional groups that actively participate in the catalytic process.

Here, we describe how SAC is applicable to guanine nucleotide-binding proteins (G proteins) in two different aspects: 1) naturally occurring SAC uses GTP as a general base in the GTPase reaction catalyzed by G proteins. 2) 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. We use a novel structure superimposition approach to analyze the anatomy of different G-proteins' active sites and the role of specific residues in the GTPase mechanism. We also discuss why the role of G-proteins as molecular switches evolved catalytic inefficiency. We describe the implications for the catalytic mechanism and how SAC paves the way to designing novel anti-cancer drugs to restore the blocked GTPase reaction that drives many human cancers.

substrate assisted catalysis: implications for drug design  

Substrate-Assisted Catalysis: Implications for Biotechnology and Drug Design.

Mickey Kosloff, Tsaffrir Zor and Zvi Selinger
Drug Dev. Res. (2000) 50: 250-257.
web pdf (135 Kb)

In Substrate-Assisted Catalysis (SAC) the substrate for enzymatic catalysis provides one or more functional groups that actively participate in the catalytic process.

Here, we describe the occurrence of SAC in natural enzymes and its use as an engineered tool to study catalytic mechanisms. We explain how this paves the way to novel therapeutic and biotechnological approaches aimed at restoring the activity of mutant inactive enzymes.