4emn
Crystal structure of RpfB catalytic domain in complex with benzamidine
Structural highlights
FunctionRPFB_MYCTU Factor that stimulates resuscitation of dormant cells. Has peptidoglycan (PG) hydrolytic activity. Active in the pM concentration range. Has little to no effect on actively-growing cells. PG fragments could either directly activate the resuscitation pathway of dormant bacteria or serve as a substrate for endogenous Rpf, resulting in low molecular weight products with resuscitation activity.[1] [2] [3] [4] Reduces lag phase and enhances the growth of quiescent (1 month-old culture) M.tuberculosis; works best between 8 and 128 pM. Increases the number of bacteria that can be recovered from a 3 month-old culture. Stimulates growth of stationary phase M.bovis (a slowly-growing Mycobacterium) as well as M.smegmatis cells (a fast grower). Binds N,N',N-triacetylchitotriose (tri-NAG). A fragment (residues 194-362) hydrolyzes an artificial lysozyme substrate 4-methylumbelliferyl-beta-D-N,N',N-triacetylchitotrioside (MUF tri-NAG). By itself has little activity on cell wall, in combination with RipA is active against cell wall extracts from a number of Actinobacteria; this activity is inhibited by PBP1A (ponA1). Sequential gene disruption indicates RpfB and RpfE are higher than RpfD and RpfC in functional hierarchy.[5] [6] [7] [8] Publication Abstract from PubMedInactivation of revival of Mycobacterium tuberculosis from dormancy is one of the main goals of the WHO Global Plan to stop tuberculosis (TB) 2011-2015, given the huge reservoir of latently infected individuals. This process requires a group of secreted proteins, denoted as resuscitation-promoting factors (Rpfs). Of these, RpfB is the sole member indispensable for resuscitation in vivo. The first class of inhibitors of RpfB was identified among 2-nitrophenylthiocyanates. However, their inactivation mechanism is hitherto not known. To gain insight into the inactivation mechanism of one of the most promising RpfB inhibitors, 4-benzoyl-2-nitrophenyl thiocyanate, NPT7, we have performed replica exchange molecular dynamics (REMD) simulations, starting from the crystal structure of RpfB catalytic domain, derived in this study. We validated our results by resuscitation experiments of M. tuberculosis cultures. The atomic resolution crystal structure of RpfB catalytic domain identified the potential of the enzyme catalytic cleft to bind benzene rings. REMD simulations, 48 replicas, identified the key interactions for the binding of NPT7 to RpfB catalytic site. Of these, an important role is played by the thiocyanate group of NPT7. Consistently, we prove that the substitution of this group implies a complete loss of RpfB inactivation. Our results provide valuable information for modifications of NPT7 structure to enhance its binding affinity to RpfB, with the final aim of developing second-generation inhibitors of therapeutic interest in TB eradication strategy. Molecular determinants of inactivation of the resuscitation promoting factor B from Mycobacterium tuberculosis.,Ruggiero A, Marchant J, Squeglia F, Makarov V, De Simone A, Berisio R J Biomol Struct Dyn. 2013;31(2):195-205. doi: 10.1080/07391102.2012.698243. Epub , 2012 Jul 25. PMID:22831279[9] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. References
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