Identification, modeling, and characterization studies of Tetrahymena thermophila myosin FERM domains suggests a conserved core fold but functional differences.

Myosins (MYO) define a superfamily of motor proteins which facilitate movement along cytoskeletal actin filaments in an ATP-dependent manner. To date, over 30 classes of myosin have been defined that vary in their roles and distribution across different taxa. The multidomain tail of myosin is responsible for the observed functional differences in different myosin classes facilitating differential binding to different cargos. One domain found in this region, the FERM domain, is found in several diverse proteins and is involved in many biological functions ranging from cell adhesion and actin-driven cytoskeleton assembly to cell signaling. Recently, new classes of unconventional myosin have been identified in Tetrahymena thermophila. In this study, we have identified, modeled, and characterized eight FERM domains from the unconventional T. thermophila myosins as their complete functional MyTH4-FERM cassettes. Our results reveal notable sequence, structural, and electrostatic differences between T. thermophila and other characterized FERM domains. Specifically, T. thermophila FERM domains contain helical inserts or extensions, which contribute to significant differences in surface electrostatic profiles of T. thermophila myosin FERMs when compared to the conventional FERM domains. Analyses of the modeled domains reveal differences in key functional residues as well as phosphoinositide-binding signatures and affinities. The work presented here broadens the scope of our understanding of myosin classes and their inherent functions, and provides a platform for experimentalists to design rational experimental studies to test the functional roles for T. thermophila myosins.

Molecular modeling and computational analyses suggests that the Sinorhizobium meliloti periplasmic regulator protein ExoR adopts a superhelical fold and is controlled by a unique mechanism of proteolysis.

The Sinorhizobium meliloti periplasmic ExoR protein and the ExoS/ChvI two-component system form a regulatory mechanism that directly controls the transformation of free-living to host-invading cells. In the absence of crystal structures, understanding the molecular mechanism of interaction between ExoR and the ExoS sensor, which is believed to drive the key regulatory step in the invasion process, remains a major challenge. In this study, we present a theoretical structural model of the active form of ExoR protein, ExoRm , generated using computational methods. Our model suggests that ExoR possesses a super-helical fold comprising 12 α-helices forming six Sel1-like repeats, including two that were unidentified in previous studies. This fold is highly conducive to mediating protein-protein interactions and this is corroborated by the identification of putative protein binding sites on the surface of the ExoRm protein. Our studies reveal two novel insights: (a) an extended conformation of the third Sel1-like repeat that might be important for ExoR regulatory function and (b) a buried proteolytic site that implies a unique proteolytic mechanism. This study provides new and interesting insights into the structure of S. meliloti ExoR, lays the groundwork for elaborating the molecular mechanism of ExoRm cleavage, ExoRm -ExoS interactions, and studies of ExoR homologs in other bacterial host interactions.

Inactivation of tesA reduces cell wall lipid production and increases drug susceptibility in mycobacteria.

Phthiocerol dimycocerosates (PDIMs) and phenolic glycolipids (PGLs) are structurally related lipids noncovalently bound to the outer cell wall layer of Mycobacterium tuberculosis, Mycobacterium leprae, and several opportunistic mycobacterial human pathogens. PDIMs and PGLs are important effectors of virulence. Elucidation of the biosynthesis of these complex lipids will not only expand our understanding of mycobacterial cell wall biosynthesis, but it may also illuminate potential routes to novel therapeutics against mycobacterial infections. We report the construction of an in-frame deletion mutant of tesA (encoding a type II thioesterase) in the opportunistic human pathogen Mycobacterium marinum and the characterization of this mutant and its corresponding complemented strain control in terms of PDIM and PGL production. The growth and antibiotic susceptibility of these strains were also probed and compared with the parental wild-type strain. We show that deletion of tesA leads to a mutant that produces only traces of PDIMs and PGLs, has a slight growth yield increase and displays a substantial hypersusceptibility to several antibiotics. We also provide a robust model for the three-dimensional structure of M. marinum TesA (TesAmm) and demonstrate that a Ser-to-Ala substitution in the predicted catalytic Ser of TesAmm renders a mutant that recapitulates the phenotype of the tesA deletion mutant. Overall, our studies demonstrate a critical role for tesA in mycobacterial biology, advance our understanding of the biosynthesis of an important group of polyketide synthase-derived mycobacterial lipids, and suggest that drugs aimed at blocking PDIM and/or PGL production might synergize with antibiotic therapy in the control of mycobacterial infections.

Identification and structural characterization of FYVE domain-containing proteins of Arabidopsis thaliana.

BACKGROUND: FYVE domains have emerged as membrane-targeting domains highly specific for phosphatidylinositol 3-phosphate (PtdIns(3)P). They are predominantly found in proteins involved in various trafficking pathways. Although FYVE domains may function as individual modules, dimers or in partnership with other proteins, structurally, all FYVE domains share a fold comprising two small characteristic double-stranded beta-sheets, and a C-terminal alpha-helix, which houses eight conserved Zn2+ ion-binding cysteines. To date, the structural, biochemical, and biophysical mechanisms for subcellular targeting of FYVE domains for proteins from various model organisms have been worked out but plant FYVE domains remain noticeably under-investigated. RESULTS: We carried out an extensive examination of all Arabidopsis FYVE domains, including their identification, classification, molecular modeling and biophysical characterization using computational approaches. Our classification of fifteen Arabidopsis FYVE proteins at the outset reveals unique domain architectures for FYVE containing proteins, which are not paralleled in other organisms. Detailed sequence analysis and biophysical characterization of the structural models are used to predict membrane interaction mechanisms previously described for other FYVE domains and their subtle variations as well as novel mechanisms that seem to be specific to plants. CONCLUSIONS: Our study contributes to the understanding of the molecular basis of FYVE-based membrane targeting in plants on a genomic scale. The results show that FYVE domain containing proteins in plants have evolved to incorporate significant differences from those in other organisms implying that they play a unique role in plant signaling pathways and/or play similar/parallel roles in signaling to other organisms but use different protein players/signaling mechanisms.

Proteomic tools for the analysis of cytoskeleton proteins.

Proteomic tools have become an essential part of the tool kit of the molecular biologist, and provide techniques for detecting homologous sequences, recognizing functional domains, modeling, and analyzing the three-dimensional structure for any given protein sequence. Although a wealth of structural and functional information is available for a large number of members of the various classes of cytoskeletal proteins, many more members remain uncharacterized. These computational tools that are freely and easily accessible to the scientific community provide an excellent starting point to predict the structural and functional properties of such partially or fully uncharacterized protein sequences, and can lead to elegantly designed experiments to probe the hypothesized function. This chapter discusses various proteomic analysis tools with a focus on protein structure and function predictions.

Molecular modeling of the membrane targeting of phospholipase C pleckstrin homology domains.

Phospholipases C (PLCs) reversibly associate with membranes to hydrolyze phosphatidylinositol-4, 5-bisphosphate (PI[4,5]P(2)) and comprise four main classes: beta, gamma, delta, and epsilon. Most eukaryotic PLCs contain a single, N-terminal pleckstrin homology (PH) domain, which is thought to play an important role in membrane targeting. The structure of a single PLC PH domain, that from PLCdelta1, has been determined; this PH domain binds PI(4,5)P(2) with high affinity and stereospecificity and has served as a paradigm for PH domain functionality. However, experimental studies demonstrate that PH domains from different PLC classes exhibit diverse modes of membrane interaction, reflecting the dissimilarity in their amino acid sequences. To elucidate the structural basis for their differential membrane-binding specificities, we modeled the three-dimensional structures of all mammalian PLC PH domains by using bioinformatic tools and calculated their biophysical properties by using continuum electrostatic approaches. Our computational analysis accounts for a large body of experimental data, provides predictions for those PH domains with unknown functions, and indicates functional roles for regions other than the canonical lipid-binding site identified in the PLCdelta1-PH structure. In particular, our calculations predict that (1). members from each of the four PLC classes exhibit strikingly different electrostatic profiles than those ordinarily observed for PH domains in general, (2). nonspecific electrostatic interactions contribute to the membrane localization of PLCdelta-, PLCgamma-, and PLCbeta-PH domains, and (3). phosphorylation regulates the interaction of PLCbeta-PH with its effectors through electrostatic repulsion. Our molecular models for PH domains from all of the PLC classes clearly demonstrate how a common structural fold can serve as a scaffold for a wide range of surface features and biophysical properties that support distinctive functional roles.