Characterizing CwsA and CrgA interaction in a lipid bilayer with ssNMR

Rongfu Zhang, Huajun Qin, Riqiang Fu, Yan-Yan Hu, Tim Cross

Florida State University, National High Magnetic Field Laboratory

Tuberculosis (TB) is a leading infectious disease killer worldwide caused by Mycobacterium tuberculosis (Mtb). CwsA and CrgA are both small integral membrane proteins that belong to Mtb cell divisome system. Previous studies by Plocinski et al. showed that CwsA interacts with CrgA, and the disruption of this interaction results in the abnormal elongation at the poles and division at mid-cell region. It is therefore important to understand this interaction at an atomic-level resolution. However, very few techniques are suitable for this challenging question, and solid-state NMR has been proven to be effective for such a heterogenous system.  We report here that this interaction can be characterized through both oriented (OS) and magic angle spinning (MAS) ssNMR approaches. We first determined the tilt of a carefully designed CwsA transmembrane fragment in a POPC/POPG (4/1) lipid bilayer environment with glass slides supported OS ssNMR. The tilt of this CwsA transmembrane domain was fitted to be ~25°. The inter-helical distance restraints between CwsA CrgA complex were obtained through dipolar assisted rotational resonance (DARR) pulse sequence on different isotope specific labeled CwsA and CrgA, and several unambiguous distances were obtained. CrgA topology in a lipid bilayer was previously characterized by Nabnita et al.  Together, a schematic model of CwsA/CrgA transmembrane domain complex was presented. Further molecular dynamic simulation can be used to improve the accuracy of this model. The structural model of CwsA and CrgA interaction improves our understanding of Mtb cell division machinery.