The structure of the N‐terminal module of the cell wall hydrolase RipA and its role in regulating catalytic activity

Abstract

RipA plays a vital role during cell division of Mycobacterium tuberculosis by degrading the cell wall peptidoglycan at the septum, allowing daughter cell separation. The peptidoglycan degrading activity relies on the NlpC/P60 domain, and as it is potentially harmful when deregulated, spatial and temporal control is necessary in this process. The N-terminal domain of RipA has been proposed to play an inhibitory role blocking the C-terminal NlpC/P60 domain. Accessibility of the active site cysteine residue is however not limited by the presence of the N-terminal domain, but by the lid-module of the inter-domain linker, which is situated in the peptide binding groove of the crystal structures of the catalytic domain. The 2.2 Å resolution structure of the N-terminal domain, determined by Se-SAD phasing, reveals an all-α-fold with 2 long α-helices, and shows similarity to bacterial periplasmic protein domains with scaffold-building role. Size exclusion chromatography and SAXS experiments are consistent with dimer formation of this domain in solution. The SAXS data from the periplasmic two-domain RipA construct suggest a rigid baton-like structure of the N-terminal module, with the catalytic domain connected by a 24 residue long flexible linker. This flexible linker allows for a catalytic zone, which is part of the spatiotemporal control of peptidoglycan degradation.

A high-flux automated laboratory small-angle X-ray scattering instrument optimized for solution scattering

“…In the application examples, the scattering from a standard Lyz protein sample showed a monomeric protein form factor with no significant structure factor contribution. The data, binned to be approximately equidistant on a logarithmic q scale, were fitted with the monomer protein crystal structure 1lyz (Diamond, 1974) using CRYSOL (Svergun et al, 1995) from the ATSAS package (Franke et al, 2017) (default settings, no constant added, Version 2.8.3) to 2 = 0.77, and using the in-house WLSQ_PDBx software (Steiner et al, 2018) (constant added) to 2 = 0.84, confirming the expected monomer protein structure (Fig. 6).…”

“…Additional studies with solution scattering data from the flux-optimized HyperSAXS laboratory instrument have been published, highlighting the capabilities of the instrument (van 't Hag et al, 2016;Manuguerra et al, 2017;Maric et al, 2017;Steiner et al, 2018;Gounani et al, 2019;Poghosyan et al, 2019;Najarzadeh et al, 2019;Pedersen et al, 2019;Nagaraj et al, 2020;Fehé r et al, 2020).…”

Complementary substrate specificity and distinct quaternary assembly of the Escherichia coli aerobic and anaerobic β-oxidation trifunctional enzyme complexes

“…The output from GNOM was further used to generate the ab initio shapes using DAMMIN of the online-SAXS cluster at EMBL, Hamburg [35,36] and for calculating low resolution electron density maps using the DENSSWeb server with the default parameters [37]. The built atomic models were compared against the experimental SAXS data using FoXS [38], CRYSOL [39] and WLSQ_PDBX [40], which also allows inclusion of the scattering from C12E9 micelles as an extra component to enable description of samples that may have coexisting micelles. The reduced  2 values were calculated and used for assessing the quality of the fits.…”

The Cell Wall Hydrolytic NlpC/P60 Endopeptidases in Mycobacterial Cytokinesis: A Structural Perspective

“…In RipB, the pro-domain is folded in two helices wrapped around the catalytic domain, suggesting a similar regulatory function as observed for RipA [51]. Compared to RipB, RipA contains an extra N-terminal domain, whose structure has been recently determined [50] (PDB code 6ewy). This domain is formed by two helices of similar length, connected by a 6-residue loop, and forms a long-coiled coil structure (Figure 6).…”

“…Instead, its rigid stalk-like module is typical of scaffold building proteins. Possibly, this non-catalytic domain is responsible for anchoring the enzyme to the divisome, although interactions of RipA with the divisome have so far not been demonstrated [50]. Different from RipA, the homologue NlpC/P60 endopeptidase RipC interacts with FtsX [57], and this interaction favors a long-range conformational change that activates RipC [15,57] (Figure 7).…”