Demonstration of molecular interactions between the murein polymerase PBP1B, the lytic transglycosylase MltA, and the scaffolding protein MipA of Escherichia coli

Enlargement of the stress-bearing murein sacculus of bacteria depends on th e coordinated interaction of murein synthases and hydrolases. To understand the mechanism of interaction of these two classes of proteins affinity chr omatography and surface plasmon resonance (SPR) studies were performed. The membrane-bound lytic transglycosylase MltA when covalently linked to CNBr- activated Sepharose specifically retained the penicillin-binding proteins ( PBPs) 1B, 1C, 2, and 3 from a crude Triton X-100 membrane extract of Escher ichia coli. In the presence of periplasmic proteins also PBP1A was specific ally bound. At least five different non-PBPs showed specificity for MltA-Se pharose. The amino-terminal amino acid sequence of one of these proteins co uld be obtained, and the corresponding gene was mapped at 40 min on the E. coli genome. This MltA-interacting protein, named MipA, in addition binds t o PBP1B, a bifunctional murein transglycosylase/ transpeptidase. SPR studie s with PBP1B immobilized to ampicillin-coated sensor chips showed an oligom erization of PBP1B that may indicate a dimerization, Simultaneous applicati on of MipA and MltA onto a PBP1B sensor chip surface resulted in the format ion of a trimeric complex, The dissociation constant was determined to be a bout 10(-6) M, The formation of a complex between a murein polymerase (PBP1 B) and a murein hydrolase (MltA) in the presence of MipA represents a first step in a reconstitution of the hypothetical murein-synthesizing holoenzym e, postulated to be responsible for controlled growth of the stress-bearing sacculus of E. coli.