Role of Force Fields in Protein Function Prediction

The world today, although, has developed an elaborate health system to fortify against known and unknown diseases, it continues to be challenged by new as well as emerging, and re-emerging infectious disease threats with severity and probable fluctuations. These threats also have varying costs for morbidity and mortality, as well as for a complex set of socio-economic outcomes. Some of these diseases are often caused by pathogens which use humans as host. In such cases, it becomes paramount responsibility to dig out the source of pathogen survival to stop their population growth. Sequencing genomes has been finessed so much in the 21st century that complete genomes of any pathogen can be sequenced in a matter of days following which; different potential drug targets are needed to be identified. Structure modeling of the selected sequences is an initial step in structure-based drug design (SBDD). Dynamical study of predicted models provides a stable target structure. Results of these in-silico techniques greatly depend on force field (FF) parameters used. Thus, in this chapter, we intend to discuss the role of FF parameters used in protein structure prediction and molecular dynamics simulation to provide a brief overview on this area

<em>Plasmodium falciparum</em>: Experimental and Theoretical Approaches in Last 20 Years

Malaria, the severe vector-borne disease has embedded serious consequences on mankind since ages, causing deterioration of health, leading to deaths. The causative parasite has a wide distribution aligned from tropical to subtropical regions. Out of all the five species Plasmodium vivax and Plasmodium falciparum have registered about more than 600 million cases worldwide. Throughout the decades, identification of various antimalarial drugs, targets, preventive measures and advancement of vaccines were achieved. The key to executing malaria elimination is the appropriate laboratory diagnosis. Development includes positive scientific judgments for a vaccine, advanced progress of 3 non-pyrethroid insecticides, novel genetic technologies, possibilities to alter malaria parasite mediation by the mosquito, identification of drug resistance markers, initiation of Plasmodium vivax liver stage assessment, perspective to mathematical modeling and screening for active ingredients for drugs and insecticides. Although the last century witnessed many successful programs with scientific progress, however, this was matched with notable obstacles. The mutation in the genes has changed the overall gameplay of eradication. This chapter aims to examine the numerous experimental and theoretical works that have been established in the last two decades along with the ongoing methodologies consisting of detailed explanations necessary for the establishment of new targets and drugs

Dynamics and Architecture of the HOPS Tethering Complex in Yeast Vacuole Fusion

The evolvement of a complex endomembrane system, which is separating a variety of biochemical processes into distinct compartments, is a hallmark of eukaryotic cell development. Cellular homeostasis depends on the abilities of these lipid bilayer-enclosed organelles both to maintain distinct characteristics and to exchange materials. This is mainly achieved by a process called vesicular transport, which allows for a constant exchange of proteins, lipids and metabolites between different compartments. Lipid bilayer enclosed vesicles bud from the donor compartment, are transported to the target compartment and fuse with its surrounding membrane. The basic machineries involved in the process in budding and fusion have been intensely investigated in the last years. However, our knowledge about the processes, which confer target specificity and regulate intracellular membrane fusion, is still limited. Before fusion of two-compartments can occur, they have to specifically recognize and bind each other to allow for subsequent SNARE-induced fusion to take place. This early step in the fusion reaction is called tethering and involves the action of tethering factors and Rab GTPases. In my research, I focused on the HOPS protein complex that is implicated to function in the tethering process at the yeast vacuole, the fungal equivalent of lysosomes. To investigate the molecular properties that confer the functionality of this large hexameric complex, I established a method that allowed for the purification of substantial amounts of HOPS and investigated the interactions taking place between different subunits. This work paved the ground for electron microscopy analysis of the whole complex, which is currently performed and which yielded first, preliminary data. Furthermore, it allowed for the identification of the novel CORVET tethering complex at the endosome, which has several subunits in common with the HOPS complex. I was able to show that chimeric complexes exist, harboring both HOPS- and CORVET-specific subunits. This finding suggests that both complexes are dynamic and can interconvert. During my studies on the subunits’ interactions, I identified stable subcomplexes of the HOPS complex, for one of which I could show that it exists in vivo. The existence of such subcomplexes implies a much more dynamic functioning of the HOPS subunits than previously anticipated. This notion is further strengthened by my studies on the functionality of different subunits and subcomplexes. Intriguingly, my results show that the Rab Guanyl nucleotide exchange factor Vam6, which is needed to activate the vacuolar Rab Ypt7 for subsequent fusion and which is a component of the HOPS complex, loses its ability to interact with Ypt7 upon incorporation into a subcomplex or the fully assembled HOPS complex. In contrast to this, the subunit Vps41, which I could identify as a Rab effector, is active as a single protein and as part of the complex, suggesting that it might sequentially recruit the subcomplexes to assemble into the holo-complex at sites harboring active Ypt7. Another feature of Vps41 was addressed in my work. This protein was previously shown to be phosphorylated by the vacuolar casein kinase Yck3. I identified the phosphorylation sites in the Vps41 sequence, which allowed further studies on the effect of the phosphorylation on the functionality of the protein. In the phosphorylated state, the protein is displaced into the cytosol whereas it accumulates at endosomal-vacuolar fusion sites if phosphorylation is prevented. Intriguingly, we found that Ypt7 overexpression is able to partially rescue the loss of localization in the phosphomimetic mutant, indicating a cross-talk between these two layers of Vps41 regulation

Contribution à la caractérisation fonctionnelle de protéines de contrôle de la sécrétion d'effecteurs de type III chez la bactérie phytopathogène Ralstonia solanacearum : chaperonnes et protéine à domaine T3S4

La bactérie phytopathogène Ralstonia solanacearum est l'agent responsable du flétrissement bactérien sur plus de 200 espèces végétales, dont des espèces agronomiques, faisant de cette bactériose une des plus importantes dans le monde. Le pouvoir pathogène de la bactérie repose en grande partie sur sa capacité à injecter des protéines, appelées effecteurs de type III (ET3s), via le système de sécrétion de type III (SST3). La dernière décennie a été notamment marquée par la découverte chez les bactéries pathogènes de nombreuses protéines impliquées dans le contrôle du processus de sécrétion de type III. Chez R. solanacearum, ces mécanismes de contrôle de la sécrétion restent méconnus, contrairement aux mécanismes de régulation transcriptionnelle. Au cours de ces travaux, nous nous sommes attachés à caractériser les fonctions des protéines HpaB (RSp0853), HpaD (RSp0848) et FliT-like (RSc2897) pour lesquelles plusieurs éléments suggèrent un potentiel rôle comme chaperonnes de type III (CT3s), ainsi que de la protéine HpaP (RSp0862) qui présente un domaine T3S4 (Type III Secretion Substrate Specificity Switch). Nous avons pu mettre en évidence la capacité de certaines CT3s à interagir entre elles et, pour HpaB et HpaD, à interagir avec de nombreux ET3s. De plus, les trois CT3s putatives semblent impliquées dans le pouvoir pathogène de R. solanacearum, HpaB s'avérant même indispensable à la virulence de la bactérie. D'autre part, nos travaux mettent en exergue l'importance de la protéine HpaP pour le pouvoir pathogène de la bactérie et son implication dans le contrôle de la sécrétion de substrats du SST3. Les résultats suggèrent notamment que HpaP promeut la sécrétion de l'ET3 PopP1 en interagissant physiquement avec ce dernier. Finalement, la caractérisation de séquences conservées du domaine T3S4 révèle l'importance de cette région pour la fonction de la protéine HpaP. L'ensemble de ces travaux suggère l'implication de plusieurs protéines de R. solanacearum dans le contrôle du processus de sécrétion de type III et souligne la diversité des mécanismes mis en jeu impliquant les protéines de type T3S4 chez les bactéries pathogènes.The plant pathogenic bacterium Ralstonia solanacearum is the causative agent of the bacterial wilt on more than 200 plant species, including agronomic species, making it one of the most important bacterial disease in the world. The pathogenicity of the bacteria is largely based on its ability to inject proteins, called type III effectors (T3Es) via the type III secretion system (T3SS) . The last decade has been particularly marked by the discovery of many proteins involved in the control of the type III secretion process in pathogenic bacteria. In R. solanacearum , these control mechanisms remain unknown , unlike the transcriptional regulatory mechanisms. In this work, we focused on the functional characterization of the proteins HpaB (Rsp0853), HpaD (RSp0848) and FliT-like (RSc2897) for which several elements suggest a potential role as type III chaperones (T3Cs). We also focused on the HpaP protein (Rsp0862) which harbors a T3S4 domain (Type III Secretion Substrate Specificity Switch). We showed the ability of some CT3s to interact with each other and, concerning HpaB and HpaD, to interact with many T3Es. In addition, the three putative T3Cs seem to be involved in the pathogenicity of R. solanacearum, HpaB being even strictly required for bacterial virulence. Furthermore, our work highlights the importance of HpaP in pathogenicity and its involvement in the control of the secretion of T3SS substrates. The results suggest in particular that HpaP promotes the secretion of the T3E PopP1 by physically interacting with the latter. Finally, the characterization of conserved sequences in the T3S4 domain reveals the importance of this region for the function of the HpaP protein. On the whole, this work suggests the involvement of several proteins of R. solanacearum in the control of the type III secretion process and highlights the diversity of mechanisms in which T3S4 proteins are involved in pathogenic bacteria

Control of Type III-mediated Virulence in Pseudomonas syringae by Cyclic-di-GMP

Pseudomonas syringae is a prominent plant pathogen and disease model organism. These bacteria carry out host infection using the type III secretion system (T3SS), which translocates effector proteins into target cells, altering cellular defence mechanisms and metabolism to promote bacterial colonisation. It was previously shown that the secondary signalling molecule cyclic-di-GMP (CdG) binds to the export ATPase complex at the base of the T3SS (HrcN in P. syringae), as well as in closely related homologue proteins. It was hypothesised that this binding interaction plays a role in controlling type III-mediated virulence. To investigate the CdG:HrcN binding interaction, bacterial strains carrying mutations targeting key predicted CdG binding residues in HrcN were constructed. In vitro analyses of purified HrcN confirmed CdG binding and dodecamerisation for the wildtype. However, a G176A point-mutant of HrcN retained CdG binding but appeared to have compromised CdG-induced downstream oligomerisation. The effect of this mutation on virulence was therefore explored in vivo. Wildtype and mutant hrcN P. syringae pathovar tomato (Pto) DC3000 strains were infiltrated into Arabidopsis thaliana to evaluate disease phenotypes in planta. The G176A hrcN point-mutant exhibited a near-asymptomatic disease phenotype despite having a comparable bacterial load to the WT in A. thaliana Col-0. Disease symptoms returned in immunocompromised A. thaliana lines. The underlying mechanism was then explored. It was shown that a subset of tested effectors (HopAM1, HopAF1, and HopAA1-2) displayed compromised translocation rates for G176A hrcN compared to WT using an effector-CyaA reporter system in Pto. These effector proteins were shown to be important for disease symptom establishment by way of gene over-expression and gene deletion. Candidate interaction targets in the plant host were identified by co-immunoprecipitation. From this study, first indication that CdG binding to HrcN in Pto may lead to dodecamerisation was shown, and that this interaction is important for full virulence by enabling for efficient translocation of key effector proteins

Identification and analysis of structurally critical fragments in HopS2

Abstract Background Among the diverse roles of the Type III secretion-system (T3SS), one of the notable functions is that it serves as unique nano machineries in gram-negative bacteria that facilitate the translocation of effector proteins from bacteria into their host. These effector proteins serve as potential targets to control the pathogenicity conferred to the bacteria. Despite being ideal choices to disrupt bacterial systems, it has been quite an ordeal in the recent times to experimentally reveal and establish a concrete sequence-structure-function relationship for these effector proteins. This work focuses on the disease-causing spectrum of an effector protein, HopS2 secreted by the phytopathogen Pseudomonas syringae pv. tomato DC3000. Results The study addresses the structural attributes of HopS2 via a bioinformatics approach to by-pass some of the experimental shortcomings resulting in mining some critical regions in the effector protein. We have elucidated the functionally important regions of HopS2 with the assistance of sequence and structural analyses. The sequence based data supports the presence of important regions in HopS2 that are present in the other functional parts of Hop family proteins. Furthermore, these regions have been validated by an ab-initio structure prediction of the protein followed by 100 ns long molecular dynamics (MD) simulation. The assessment of these secondary structural regions has revealed the stability and importance of these regions in the protein structure. Conclusions The analysis has provided insights on important functional regions that may be vital to the effector functioning. In dearth of ample experimental evidence, such a bioinformatics approach has helped in the revelation of a few structural regions which will aid in future experiments to attain and evaluate the structural and functional aspects of this protein family