Structure/Function Analysis of Human Integral Membrane Proteins: Connexin 26 and the ZnT8 Zinc Transporter

In this dissertation I present a detailed structural and functional characterization of two human membrane proteins – zinc transporter ZnT8 and gap junction channel and hemichannel Connexin26.

The human zinc transporter ZnT8 (SLC30A8), expressed primarily in pancreatic β-cells, plays a key function in maintaining the concentration of blood glucose through its role in insulin storage, maturation and secretion. This transporter is an autoantigen for Type 1 diabetes (T1D) and is associated with Type 2 diabetes (T2D) through its risk allele that encodes a major non-synonymous single nucleotide polymorphism (SNP) at Arg325. Despite its role in diabetes and concomitant potential as a drug target, little is known about the structure or mechanism of ZnT8. To this end, we expressed ZnT8 in Pichia pastoris yeast and Sf9 insect cells. Guided by a rational screen of 96 detergents, we developed a method to solubilize and purify recombinant ZnT8, which is stable in fos-choline 12 for >2 weeks. An in vivo transport assay in Pichia showed that the protein is functionally active. A fluorescence-based in vitro transport assay using proteoliposomes indicated that human ZnT8 functions as a Zn2+/H+ antiporter in liposomes. We also purified E. coli-expressed amino- and carboxy-terminal cytoplasmic domains. Circular dichroism spectrometry showed that the amino-terminal domain contains predominantly β-sheet and turn secondary structure, and the carboxy-terminal domain has a mixed α/β structure. Electron microscopy and single-particle image analysis yielded a density map of ZnT8B at 20-Å resolution, which revealed that it forms a dimer in detergent micelles. Two prominent lobes are ascribed to the transmembrane domains, which recapitulate the architecture of the bacterial zinc transporter YiiP. These results provide a foundation for higher resolution structural studies and screening experiments to identify compounds that modulate ZnT8 activity.

Connexins (Cx) are a family of integral membrane proteins that assemble as hexameric hemichannels. Hemichannels from adjacent cells dock to form dodecameric gap junction channels (GJCs). GJCs mediate intercellular communication between adjacent cells and exhibit properties of both ligand- and voltage-gated channels, including channel closure at acidic pH. Notably, mutations of the Cx26 gene (GJB2) are the most common cause of sensorineural hearing loss in children. Despite numerous functional studies on gating of connexins, the structural basis for pH-induced gating is not well understood. In order to gain insight into this mechanism, recombinant, human Cx26 was purified as dodecameric GJCs, which remained homogenous and monodisperse when reconstituted in amphipol. To explore pH inhibition, we herein used single-particle, electron cryo-microscopy and mass spectrometry to examine the conformational changes associated with pH-mediated regulation. In pH 7.5, the structure of the Cx26 GJCs reconstituted in amphipol recapitulated the previous GJC crystal structures. Within the transmembrane and extracellular domains, the map resolution was ~3.5 Å, whereas the N-terminus showed a conformationally heterogenous state, which was confirmed by hydrogen deuterium exchange mass spectrometry. Three-dimensional classification of particles at pH 6.4 yielded two distinct conformations, one resembling the physiological pH structure and another with a pore-occluding density. Modeling of the polypeptide into the cryoEM maps suggests that acidic pH elicits an extension and association of the amino-termini to form the occluding gating particle. Lysine crosslinking with tandem mass spectrometry revealed additional crosslinks at acidic pH between residues K15 and K22 and the cytoplasmic loop, indicative of closer association compared with pH 7.5. Taken together, our results support a ‘ball-and-chain’ mechanism in which the NTs undergo an extension and associate to occlude the channel pore during pH-mediated gating.

The physiological roles of connexin hemichannels and gap junction channels can be distinguished by their characteristic regulation. GJCs serve as intercellular connections and are in a constitutively open state so that tissues function as syncytia. HCs in the plasma membrane communicate with the extracellular space and by default are in a closed state. As of the time of writing this, no structure of a connexin hemichannel exists and the mechanistic details of their regulation are not well known. To gain insight into the structure of Connexin 26 hemichannel, a mutation in connexin 26 gene was introduced (N176Y) in order to prevent connexin hemichannels from docking and forming GJCs. Electron and cryo-electron microscopy study of the N176Y hexamer in detergent and amphipol has revealed that the protein is conformationally unstable. As a consequence, the protein was reconstituted in lipid nanodiscs. CryoEM analysis of the nanodiscs on C-flat carbon grids resulted in 2D class averages clearly showing en face protein views with visible α-helical secondary structure. Unfortunately, particles displayed a preferred orientation on the grids. To remedy this, pegylated gold grids were prepared in-house, on which the protein adopted a more random orientation which resulted in 2D class averages showing both en face views and side views with traces of secondary structure. To conclude, a preparation of connexin 26 hemichannel was optimized for future high-resolution structure determination with cryoEM.