Interface integrity in septin protofilaments is maintained by an arginine residue conserved from yeast to man

The septins are conserved, filament-forming, guanine nucleotide binding cytoskeletal proteins. They assemble into palindromic protofilaments which polymerize further into higher-ordered structures that participate in essential intracellular processes such as cytokinesis or polarity establishment. Septins belong structurally to the P-Loop NTPases but, unlike their relatives Ras or Rho, do not mediate signals to effectors through GTP binding and hydrolysis. Biochemical approaches addressing how and why septins utilize nucleotides are hampered by the lack of nucleotide free complexes. Using molecular dynamics simulations, we determined inter-subunit binding free energies in human and yeast septin dimer structures and in their in silico generated apo forms. Coordination of the nucleotide by a conserved arginine in the septin unique β-meander, Arg(βb), was identified as a stabilizing element. Removal of GDP resulted in disturbance of an extensive hydrogen bond network around the guanine ring, concomitant with decreased inter-subunit affinity. We confirmed our in silico approach  in vitro and in vivo by investigating the consequences of an exchange of Arg(βb) to Ala.  Purified septins bearing a Arg(βb)(Ala) mutation did not assemble into dimers. SEPT9(RβbA) did not complement the phenotype of a SEPT9 knock out fibroblast cell line whereas SEPT9 WT does.