Figure 1: The BR domain comprises a furin protease recognition site and forms irreversibly associated dimers only during heterologous expression.
(A) PsrP is organized into five domains: the N-terminal signal sequence (S) for extracellular translocation of PsrP, two putatively glycosylated serine rich repeat regions (SRR1 and SRR2), the binding region (BR) domain and the cell wall domain (CW). BR harbors two distinct sub-regions for KRT10 binding (black bar, residues 273–341) and self-oligomerization (black bar, residues 122–166), respectively. The sequence K164RRKR168 is recognized by the human Furin protease. The three constructs BR120–395, BR187–385 and BR187–378 used within this study are displayed below the overall schematic representation of PsrP. In the mutated version of BR120–395 (BR*120–395), KRRKR was substituted to KSRKS. (B) A protease cleavage assay confirmed that furin recognized the KRRKR motif, but did not cleave in the presence of the furin protease inhibitor or when the KRRKR sequence was substituted to KSRKS. Cleavage was performed at room temperature (RT) and 37 °C for the indicated incubation times. The inhibitor was used at 100 μM and 250 μM, shown as plus signs in regular and bold format, respectively. (C) Distinct populations of BR187–385 were eluted using Superdex 200 HiLoad 16/600 at elution column volumes (CV) of 89, 78, 71 and 66 mL corresponding to apparent molecular weights of 22, 55 and 90, 150 kDa, as well as higher oligomers. In this study, we show that the irreversibly associated dimer of BR187–385 is formed through a domain swap mechanism (PDB: 5JUI, Fig. S2) and that a low-affinity β-sheet dimer is created between two symmetry-related molecules in the previously determined crystal structure of the BR187–385 monomer (PDB: 3ZGH, Fig. 3). (D) BR*120–395 was eluted from the same column at CV of 79 and 69 mL corresponding to apparent MW of 50 and 115 kDa. (C,D) When the isolated monomer and oligomer populations of BR187–385 and BR*120–395 were re-applied on analytical Superdex 200 HR10/30 columns, the monomer and oligomer populations were stable and did not interconvert between each other.
