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Since CpxP has no homologues of known function, we have initially focused on its biophysical and structural characterization. Using multi-angle laser light scattering (MALLS), small-angle Xray scattering (SAXS) analysis, and formaldehyde-mediated crosslinking experiments, we show that full-length E. coli CpxP is a dimer in vivo as well as in pathway inactivating (pH 5.8) and activating (pH 8.0) conditions in vitro. Far-UV circular dichroism (CD) was used to demonstrate that CpxP is mainly α-helical, while near-UV CD and SAXS revealed that the protein may undergo a small structural adjustment in response to a pathway-inducing stimulus (pH 8.0). The crystal structure of CpxP, determined to 2.85 Å resolution, revealed an antiparallel dimer of intertwined α-helices with a highly basic concave surface. Each protomer consists of a long, hooked and bent hairpin fold with conserved LTXXQ motifs forming two diverging turns at one end. Three of six previously characterized cpxP loss-of-function mutations, M 59 T, Q 55 P, and Q 128 H, likely result from a destabilization of the protein fold, whereas the R 60 Q, D 61 E, and D 61 V mutations may alter interactions important for the signalling or proteolytic adaptor functions of CpxP.

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