Design, Function and Structure of a Monomeric CLC Transporter

Abstract

The CLC family of Cl- channels and Cl-/H+ antiporters are homodimeric in structure with each subunit containing a distinct transport pathway. Yet, the role of this conserved dimeric architecture in relation to protein function is not well understood. While certain CLC channels demonstrate dimer-dependent gating through cytosolic domains, the near-normal function of a bacterial CLC transporter strait-jacketed by covalent crosslinks across the dimer interface argues that the transport cycle resides within each subunit and does not require rigid-body rearrangements between subunits. A prediction that follows is that it should be possible to construct a monomeric form of a CLC protein while preserving structural and functional properties. To investigate this, we designed a stable monomeric variant of the E. coli CLC transporter, CLC-ec1, by introducing two tryptophan mutations, I201W and I422W, at the dimer interface. The existence of monomer was confirmed by gel filtration and glutaraldehyde cross-linking in both detergent micelles and PC/PG liposomes. The monomer is functional, exhibiting H+ coupled Cl- transport at rates comparable to the wild-type dimer and with 2:1 stoichiometry. The structure of the monomer was solved to 3.1 A resolution showing a crystal packing arrangement that exposes the previously buried dimer interface. The backbone atoms of this new structure aligns with the original CLC-ec1 crystal structure with 0.4 A RMSD, showing that the intrinsic subunit structure is not affected by these mutations or separation from the dimer state. These results demonstrate that the CLC subunit alone is the basic functional unit for transport and that cross-subunit interaction is not required for Cl-/H+ exchange in CLC transporters.