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

Abstract

Carboxysomes are compartments in bacterial cells that promote efficient carbon fixation by sequestering RubisCO and carbonic anhydrase within a protein shell that impedes CO₂ 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_S, joined by unstructured linkers. CcmM_S 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 <i>Thermosynechococcus elongatus</i> CcmM_S domain, revealing that it adopts a compact, well-defined structure that resembles that of RbcS. CcmM_S, however, lacked key RbcS RubisCO-binding determinants, most notably an extended N-terminal loop. Nevertheless, individual CcmM_S domains are able to bind RubisCO <i>in vitro</i> with 1.16 μm affinity. Two or four linked CcmM_S domains did not exhibit dramatic increases in this affinity, implying that short, disordered linkers may frustrate successive CcmM_S 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_S domains can bind a single RubisCO holoenzyme and, moreover, that RbcS is not released from these complexes. CcmM_S bound equally tightly to a RubisCO variant in which the α/β domain of RbcS was deleted, suggesting that CcmM_S binds RubisCO independently of its RbcS subunit. We propose that, instead, the electropositive CcmM_S may bind to an extended electronegative pocket between RbcL dimers.