Mycobacterium tuberculosis RpfE crystal structure reveals a positively charged catalytic cleft

Abstract

Resuscitation promoting factor (Rpf) proteins, which hydrolyze the sugar chains in cellwall peptidoglycan (PG), play key roles in prokaryotic cell elongation, division, and escape from dormancy to vegetative growth. Like other bacteria, Mycobacterium tuberculosis (Mtb) expresses multiple Rpfs, none of which is individually essential. This redundancy has left unclear the distinct functions of the different Rpfs. To explore the distinguishing characteristics of the five Mtb Rpfs, we determined the crystal structure of the RpfE catalytic domain. The protein adopts the characteristic Rpf fold, but the catalytic cleft is narrower compared to Mtb RpfB. Also in contrast to RpfB, in which the substrate-binding surfaces are negatively charged, the corresponding RpfE catalytic pocket and predicted peptide-binding sites are more positively charged at neutral pH. The complete reversal of the electrostatic potential of the substrate-binding site suggests that the different Rpfs function optimally at different pHs or most efficiently hydrolyze different micro-domains of PG. These studies provide insights into the molecular determinants of the evolution of functional specialization in Rpfs.

The RpfC (Rv1884) atomic structure shows high structural conservation within the resuscitation-promoting factor catalytic domain

“…1c). The variation in surface charge between RpfB and RpfE has previously been noted (Mavrici et al, 2014). RpfC has two lysines, Lys26 and Lys33, on one side of the sugar-binding cleft, which are tyrosines in RpfA, RpfB and RpfD or a leucine in RpfE and serine or threonine in RpfA, RpfD and RpfE or an aspartate in RpfB (Fig.…”

“…2a and 2b). Compared with the recent RpfE structure (PDB entry 4cge; Mavrici et al, 2014), the calculated C r.m.s.d. is even lower at 0.82 Å for 77 residues with 62% sequence identity (Figs.…”

“…2c). Mavrici et al (2014) Arg126 may play a role in binding the peptide part of the peptidoglycan, conferring specificity on RpfE.…”

“…Is specificity based on small changes within the RPF catalytic domain structure itself or on the domain organization? The solution structure (Cohen-Gonsaud et al, 2005) and various X-ray structures of RpfB (Ruggiero et al, 2009Squeglia et al, 2013) and, very recently, the structure of RpfE have been published (Mavrici et al, 2014). In this paper, we describe the X-ray structure of the RpfC catalytic domain.…”

Lytic transglycosylases: concinnity in concision of the bacterial cell wall

“…Additionally, the endolysin of bacteriophage ΦKZ GP144 (1H) is a member of Family 1. Two proteins from Mycobacterium tuberculosis , resuscitation-promoting factor B and E, give structural evidence in support of their identification as LTs, but formation of the LT reaction product has not yet been shown (Squeglia et al , 2013; Mavrici et al , 2014; Sexton et al , 2015; Ruggiero et al , 2016). Family 1 is a diverse family in both sequence (Figure 4) and function (Lee et al , 2013).…”