Membrane Association of the Intrinsically Disordered N-Terminal Region of ChiZ

Abstract

Multiple proteins including ChiZ that comprise the cell division machinery (divisome) of Mycobacterium tuberculosis (Mtb; causative agent of tuberculosis) contain disordered regions, often enriched in cationic residues. Given the highly anionic lipid composition, membrane association of Mtb divisome proteins may be expected to play important roles in cell division, a process with implications for both pathogenesis and drug resistance. Here we combined NMR spectroscopy and molecular dynamics (MD) simulations to characterize the membrane association of ChiZ. The 165-residue ChiZ consists of a disordered cytoplasmic N-terminal region (NT; residues 1-64), a transmembrane helix (TM; residues 64-86), and a periplasmic LysM domain (residues 114-165). 1H-15N HSQC spectra showed that NT associated only with highly anionic membranes (7:3 POPG/POPE liposomes), with terminal residues remaining dynamic. 13C-13C INEPT and CP experiments further revealed that most residues, but not some arginines, were dynamic when NT was membrane-bound. PRE experiments indicated that arginines strongly interacted with lipids, both in NT and in full-length ChiZ. Extensive MD simulations found that not all arginines interacted with membranes to the same extent. NT was anchored to membranes in the midsection by Arg37, forming on average 0.93 hydrogen bonds (HBs); arginines forming a least 0.5 HBs extended upstream to Arg33 and downstream to Arg49. When tethered to membranes by TM, the above pattern was largely preserved, except now the TM-proximal Arg62 formed 1.47 HBs. Overall, the second half of NT is more adaptive to membrane association, becoming more compact, whereas the first half is more autonomous. This difference is largely attributable to the exclusive occurrence of anionic residues in the first half of the NT sequence. These insights into sequence-conformation-interaction relations have broad significance, as numerous membrane proteins may involve membrane association of disordered regions in their functional processes.