The small RbcS-like domains of the β-carboxysome structural protein CcmM bind RubisCO at a site distinct from that binding the RbcS subunit.

Carboxysomes are compartments in bacterial cells that promote efficient carbon fixation by sequestering RubisCO and carbonic anhydrase within a protein shell that impedes CO escape. The key to assembling this protein complex is CcmM, a multidomain protein whose C-terminal region is required for RubisCO recruitment. This CcmM region is built as a series of copies (generally 3-5) of a small domain, CcmM, joined by unstructured linkers. CcmM domains have weak, but significant, sequence identity to RubisCO's small subunit, RbcS, suggesting that CcmM binds RubisCO by displacing RbcS. We report here the 1.35-Å structure of the first CcmM domain, revealing that it adopts a compact, well-defined structure that resembles that of RbcS. CcmM, however, lacked key RbcS RubisCO-binding determinants, most notably an extended N-terminal loop. Nevertheless, individual CcmM domains are able to bind RubisCO with 1.16 μm affinity. Two or four linked CcmM domains did not exhibit dramatic increases in this affinity, implying that short, disordered linkers may frustrate successive CcmM domains attempting to simultaneously bind a single RubisCO oligomer. Size-exclusion chromatography-coupled right-angled light scattering (SEC-RALS) and native MS experiments indicated that multiple CcmM domains can bind a single RubisCO holoenzyme and, moreover, that RbcS is not released from these complexes. CcmM bound equally tightly to a RubisCO variant in which the α/β domain of RbcS was deleted, suggesting that CcmM binds RubisCO independently of its RbcS subunit. We propose that, instead, the electropositive CcmM may bind to an extended electronegative pocket between RbcL dimers.