RESUMEN
Acinetobacter baumannii is one of the multi-drug-resistant pathogens responsible for hospital-acquired infections reported worldwide. Clinically it is challenging to treat these pathogens as they have developed resistance against the existing class of antibiotics. Hence, there is an urgent need to develop a new class of antibiotics against these pathogens to prevent the spread of infections and mortality. In Acinetobacter baumannii, the filamentous temperature-sensitive mutant Z protein polymerizes at the imminent division site to form a Z-ring at the mid-point of the cell and act as a scaffold to recruit other cell division proteins involved in orchestrating septum synthesis in bacteria. Perturbation in the assembly of FtsZ affects bacterial cell dynamics and survival. Hence, FtsZ has emerged as a new drug target in antibiotic discovery to identify compounds that inhibit bacterial cell division. In this study, we have performed a virtual screening of 30,000 compounds from the ZINC Biogenic natural compound library targeting the nucleotide-binding site of FtsZ from Acinetobacter baumannii. We have identified 8 new natural compounds with binding energy in the range of -8.66 to -6.953 kcal/mol and analyzed them by 200 ns molecular dynamics simulations. Out of these eight compounds, ZINC14708526 showed the best binding with relatively optimal drug-likeness and medicinal chemistry as a potent inhibitor of abFtsZ. Thus, the identified FtsZ inhibitor ZINC14708526 is a promising lead compound to develop potent antimicrobial agents against Acinetobacter baumannii infections.Communicated by Ramaswamy H. Sarma.
RESUMEN
VpsR, the master regulator of biofilm formation in Vibrio cholerae, is an atypical NtrC1 type bEBP lacking residues essential for σ54-RNAP binding and REC domain phosphorylation. Moreover, transcription from PvpsL, a promoter of biofilm biosynthesis, has been documented in presence of σ70-RNAP/VpsR/c-di-GMP complex. It was proposed that c-di-GMP and VpsR together form an active transcription complex with σ70-RNAP. However, the impact of c-di-GMP imparted on VpsR that leads to transcription activation with σ70-RNAP remained elusive, largely due to the lack of the structure of VpsR and knowledge about c-di-GMP:VpsR interactions. In this direction we have solved the crystal structure of VpsRRA, containing REC and AAA+ domains, in apo, AMPPNP/GMPPNP and c-di-GMP bound states. Structures of VpsRRA unveiled distinctive REC domain orientation that leads to a novel dimeric association and noncanonical ATP/GTP binding. Moreover, we have demonstrated that at physiological pH VpsR remains as monomer having no ATPase activity but c-di-GMP imparted cooperativity to convert it to dimer with potent activity. Crystal structure of c-di-GMP:VpsRRA complex reveals that c-di-GMP binds near the C-terminal end of AAA+ domain. Trp quenching studies on VpsRR, VpsRA, VpsRRA, VpsRAD with c-di-GMP additionally demonstrated that c-di-GMP could potentially bind VpsRD. We propose that c-di-GMP mediated tethering of VpsRD with VpsRA could likely favor generating the specific protein-DNA architecture for transcription activation.
Asunto(s)
Adenosina Trifosfatasas/metabolismo , Sitios de Unión , GMP Cíclico/análogos & derivados , ADN Bacteriano/metabolismo , Adenosina Trifosfatasas/química , Proteínas Bacterianas/metabolismo , Biopelículas , Cristalografía por Rayos X , Regulación Bacteriana de la Expresión Génica , Modelos Moleculares , Fosforilación , Regiones Promotoras Genéticas , Conformación Proteica , Transducción de Señal , Vibrio cholerae/genéticaRESUMEN
Vibrio cholerae experiences a highly hostile environment at human intestine which triggers the induction of various heat shock genes. The hchA gene product of V. cholerae O395, referred to a hypothetical intracellular protease/amidase VcHsp31, is one such stress-inducible homodimeric protein. Our current study demonstrates that VcHsp31 is endowed with molecular chaperone, amidopeptidase and robust methylglyoxalase activities. Through site directed mutagenesis coupled with biochemical assays on VcHsp31, we have confirmed the role of residues in the vicinity of the active site towards amidopeptidase and methylglyoxalase activities. VcHsp31 suppresses the aggregation of insulin in vitro in a dose dependent manner. Through crystal structures of VcHsp31 and its mutants, grown at various temperatures, we demonstrate that VcHsp31 acquires two (Type-I and Type-II) dimeric forms. Type-I dimer is similar to EcHsp31 where two VcHsp31 monomers associate in eclipsed manner through several intersubunit hydrogen bonds involving their P-domains. Type-II dimer is a novel dimeric organization, where some of the intersubunit hydrogen bonds are abrogated and each monomer swings out in the opposite directions centering at their P-domains, like twisting of wet cloth. Normal mode analysis (NMA) of Type-I dimer shows similar movement of the individual monomers. Upon swinging, a dimeric surface of ~400Å2, mostly hydrophobic in nature, is uncovered which might bind partially unfolded protein substrates. We propose that, in solution, VcHsp31 remains as an equilibrium mixture of both the dimers. With increase in temperature, transformation to Type-II form having more exposed hydrophobic surface, occurs progressively accounting for the temperature dependent increase of chaperone activity of VcHsp31.