GpsB acts as an adapter for MacP-mediated activation of class A penicillin-binding protein aPBP2a in Streptococcus pneumoniae, independently of MacP phosphorylation

Regulation of class A penicillin-binding proteins (aPBPs) in peptidoglycan biosynthesis is incompletely understood in Gram-positive bacteria. One example is activation of aPBP2a by GpsB and phosphorylated MacP in the ovoid-shaped pathogen, Streptococcus pneumoniae. We set out to examine whether phosphorylation of Thr residues other than Thr32 contributed to MacP activation of aPBP2a. We also wanted to determine whether GpsB and MacP activation of aPBP2a were related. Here we report that MacP was phosphorylated about equally at Thr32 and Thr56 in physiological and biochemical assays. However, based on transformation and growth assays, phosphorylation of MacP was not required for aPBP2a activation. A structure-function analysis confirmed that most of the MacP cytoplasmic domain, which was predicted by AlphaFold3 to be disordered, was not required for aPBP2a activation. These analyses further identified amino acids in the MacP transmembrane domain and the aPBP2a juxtamembrane region, as well as a variant of the GpsB-binding motif in the membrane-proximal cytoplasmic region of MacP, required for aPBP2a activation. Together, these results support a tripartite model in which GpsB acts as an adapter for activation of aPBP2a by MacP. Finally, additional interaction, Tn-seq, and growth assays suggested other modes of direct or indirect regulation of aPBP2a activity.