Structure of CrgA,a cell division structural and regulatory protein from Mycobacterium tuberculosis,in lipid bilayers

The 93-residue transmembrane protein CrgA in Mycobacterium tuberculosis is a central component of the divisome, a large macromolecular machine responsible for cell division. Through interactions with multiple other components including FtsZ, FtsQ, FtsI (PBPB), PBPA, and CwsA, CrgA facilitates the recruitment of the proteins essential for peptidoglycan synthesis to the divisome and stabilizes the divisome. CrgA is predicted to have two transmembrane helices. Here, the structure of CrgA was determined in a liquid–crystalline lipid bilayer environment by solid-state NMR spectroscopy. Oriented-sample data yielded orientational restraints, whereas magic-angle spinning data yielded interhelical distance restraints. These data define a complete structure for the transmembrane domain and provide rich information on the conformational ensembles of the partially disordered N-terminal region and interhelical loop. The structure of the transmembrane domain was refined using restrained molecular dynamics simulations in an all-atom representation of the same lipid bilayer environment as in the NMR samples. The two transmembrane helices form a left-handed packing arrangement with a crossing angle of 24° at the conserved Gly39 residue. This helix pair exposes other conserved glycine and alanine residues to the fatty acyl environment, which are potential sites for binding CrgA’s partners such as CwsA and FtsQ. This approach combining oriented-sample and magic-angle spinning NMR spectroscopy in native-like lipid bilayers with restrained molecular dynamics simulations represents a powerful tool for structural characterization of not only isolated membrane proteins, but their complexes, such as those that form macromolecular machines.Better understanding of cell division in Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), will generate new opportunities for pharmaceutical development. CrgA, a transmembrane (TM) protein, is a central component of the Mtb divisome (1). CrgA has homologs in other actinomycetes (2, 3), but not in the two bacteria, Escherichia coli and Bacillus subtilis, with better characterized cell division mechanisms. Conversely, many cell division proteins in the latter organisms, such as FtsA, FtsN, FtsL, and ZipA, appear to have no homologs in Mtb. CrgA is localized at the poles and septum, and interacts with multiple cell division proteins, including FtsZ, FtsQ, FtsI (PBPB), PBPA, and CwsA. One function of these interactions is to stabilize the divisome (1, 4). The interaction with CwsA, a protein that is unique to mycobacteria (5), might coordinate elongation at the poles and division at midcell (4). Moreover, CrgA appears to have an important role in peptidoglycan (PG) formation during cell division, by recruiting PG synthases to the divisome (4). Reduced production of CrgA results in elongated cells and reduced growth rate (1), and loss of CrgA impairs PG synthesis (5). In addition to CwsA, the Mtb divisome involves other atypical players such as FipA (FhaB), ChiZ, and MtrB (6–8), and thus there is much yet to be learned about the participants in mycobacterial cell division (9). Here, we determined the structure of CrgA in a lipid bilayer environment using solid-state NMR (ssNMR) spectroscopy.TB is a devastating human disease that kills ∼1.3 million people each year with 8.6 million new cases diagnosed annually worldwide (10). Rising extreme drug-resistant Mtb strains do not succumb to the frontline antibiotics, generating a dire need for new drugs (11). Pathways critical for bacterial survival such as DNA replication and cell division include numerous potential drug targets and represent a major focus for structural biology. Also, TB treatment is expensive and significantly toxic and requires an extensive period caused by Mtb’s ability to exist in a latent state. Hence, there are additional motivations for characterizing the proteins associated with its survival in active and nonreplicative persistent states.CrgA was first described from Streptomyces as being required for sporulation through coordinating several aspects of its reproductive growth (2, 3). The Mtb CrgA consists of 93 residues, with two predicted TM helices (12) (TM1: residues 29–51; and TM2: residues 66–88; Fig. 1A). The N-terminal 17 residues are predicted to be disordered by the software PONDR (13); the C terminus is predicted to be just five residues, whereas the loop between the TM helices is predicted to be just 14 residues. The predicted TM1 sequence contains a pair of conserved tryptophan residues (W32 and W47) that appear from the sequence to be positioned for anchoring the helix to the membrane interfacial regions. A second pair of conserved tryptophan residues is at positions 73 and 92. Because the TM2 prediction has W73 eight residues into the helix and W92 four residues beyond the end of the predicted helix, this prediction may not be as accurate. Both predicted helices contain a number of other conserved residues, whereas the loop between the helices is much more variable both in length and in composition (SI Appendix, Fig. S1).Fig. 1.Amino acid sequence and ssNMR spectra of full-length Mtb CrgA membrane protein. (A) The sequence of the expressed CrgA with a C-terminal His₆ tag. The predicted transmembrane (TM) helical residues (TMHMM, version 2.0) are indicated by red lettering. ( ...Surprisingly, this small membrane protein binds a large number of other proteins, all of which are transmembrane proteins except for FtsZ. In particular, FtsI, with a single TM helix, is a transpeptidase responsible for synthesis of the septal PG (1). A crgA-deletion mutant results in the loss of septal and polar localization of FtsI, suggesting the importance of CrgA for PG synthesis through its recruitment of FtsI. CwsA also contains a single TM helix. A crgA and cwsA double-deletion mutant showed the importance of the corresponding gene products for cell wall synthesis and cell shape maintenance (8).The CrgA TM helices contain a number of conserved glycine and alanine residues (SI Appendix, Fig. S1). Although glycine residues are known to be helix breakers in water-soluble proteins, in TM helices, they may allow local helix bending in the low dielectric membrane environment where intrahelical hydrogen bonds are strengthened for maintaining the overall integrity of the helical structure. In addition, glycine and alanine residues permit close approach of adjacent helical backbones, resulting in backbone–backbone electrostatic and side-chain–side-chain van der Waals interactions that stabilize the tertiary structure. Therefore, glycines may allow helical membrane proteins to sacrifice secondary structural stability for tertiary structural stability (14–16). This is needed because the amino acid composition in the interior of membrane proteins is more hydrophobic than the interior of water-soluble proteins where there are more frequent tertiary hydrogen bonds than in TM domains (17). In addition, conserved glycine residues are rarely found on the fatty-acyl exposed surface of multihelix membrane proteins (16). In such a location, they would expose their hydrophilic backbone atoms to the low dielectric environment of the protein. If present, it is a strong indication that they are exposed for a required function such as binding another protein. Interestingly, E. coli FtsQ is thought to localize to the divisome through interactions with other components via its single TM helix (18).Only a couple of full-length Mtb membrane protein structures have been determined. One is an X-ray structure of the mechanosensitive channel of large conductance, and the other is a single TM helix protein, Rv1761 (19, 20). In addition, water-soluble domains of other Mtb membrane proteins have been characterized such as those from PknB and FtsX (21–24). Although X-ray crystallographers have focused on large membrane proteins, the majority of the 1,162 ORFs of the Mtb genome code for small helical membrane proteins containing one to three TM helices with <40-kDa molecular weight (25). Structure–function studies of these small membrane proteins are essential for understanding Mtb cell division and other cellular processes. Small polytopic membrane protein structures are stabilized not just by interactions between their TM helices, but also by interactions with their membrane environment. Consequently, it is necessary to solve their structures in an appropriate membrane mimetic environment, one that possesses many of the restraining influences of the native membrane such as a relatively fixed hydrophobic thickness, a dramatic lateral pressure profile, and a hydrophobic core essentially devoid of water (26, 27).For the structure determination of CrgA, here we used both oriented-sample (OS) and magic-angle spinning (MAS) ssNMR to characterize the full-length protein in lipid bilayers. All ssNMR spectroscopy was performed on fully hydrated liquid–crystalline lipid bilayer preparations of CrgA. The use of such bilayer preparations for supporting the native-like conformation of the M2 protein from Influenza A has been validated with the comparison of spectra from synthetic bilayers and from cellular membranes where the protein has been inserted by the cellular machinery and never removed from this environment or exposed to a detergent environment (28). Multiple recent membrane protein structures have now been determined by OS ssNMR (29–34), and the first membrane protein structure has been obtained from MAS ssNMR (35). OS ssNMR generates information on the orientations of peptide planes with respect to the bilayer normal, and for a TM helix it yields the tilt angle of the helix relative to the lipid bilayer normal and rotational orientation about the helical axis along the entire length of helix. However, it does not directly provide information on the helix–helix packing interface. The latter information can be ascertained by relatively few distance restraints between the helices, as the degrees of freedom for packing the helices have been minimized by the orientational restraints, to just the relative rotation around the bilayer normal and relative translation in the bilayer plane. The combination of OS and MAS ssNMR thus allows the complete determination of the helical TM domain structure.Based on the OS and MAS data, we refined the structure using restrained molecular dynamics simulations in an all-atom representation of the same lipid bilayer environment as in the protein samples. The two TM helices both have a tilt angle of 13° but are tilted in nearly opposite directions such that they form a left-handed packing arrangement with a crossing angle of 24° at the conserved Gly39 residue. The two-helix TM domain exposes other conserved glycine and alanine residues that potentially form binding sites for TM helices of CrgA binders. Much of the N-terminal region is disordered, but a nine-residue motif therein appears to form an amphipathic helix. In the interhelical loop, a short segment appears to be disordered while a 12-residue motif appears to form a β-hairpin in the membrane interface. The C terminus comprises just two residues. Overall, the structure suggests how CrgA serves as a platform where other proteins of the divisome assemble.