CryoEM Studies of KdpFABC
EMSL Project ID
50936
Abstract
Our project addresses the molecular mechanism of an unusual K+ pump called KdpFABC. In K+ deficient conditions, bacteria induce expression of this pump in order to maintain osmotic conditions necessary for cell shape and for secondary transport processes. The pump is unusual because it is a hybrid of two well-studied protein families; thus, an understanding of energy coupling and allosteric interactions will shed new light on the mechanistic properties of both families and add to our appreciation of the diverse ways in which biological organisms move substrates across biological membranes. In 2017, our group solved the first structure of the hetero-oligomeric complex of KdpFABC by X-ray crystallography. The structure showed the expected channel-like architecture of the KdpA subunit, which has been postulated to carry the ion across the membrane and the expected P-type ATPase architecture of the KdpB subunit, whose ATP-dependent conformational changes are thought to drive the transport process. In addition to auxiliary subunits KdpC and KdpF, an unprecedented tunnel was observed running through the membrane and connecting ion binding sites in KdpA and KdpB. Despite the wealth of information provided by this structure, it appears to represent an inhibited state due to a serine-phosphorylation and crystal packing constraints. We now aim to use cryo-EM to reveal the structure of reaction intermediates. To do this, we will use ligands such as K+ and analogues of ATP and PO4 to trap individual states. This approach mimics X-ray studies of the archetypal P-Type ATPase, SERCA, though without the need for crystallization. Over the last year, we have tested a variety of different preparations and cryo-EM substrates in order to arrive at a successful protocol for structure determination by cryo-EM. In particular, we have generated a double mutant of KdpFABC that 1) lowers K+ affinity to the millimolar range so that this ion can be titrated to control protein activity and 2) prevents serine phosphorylation that we have shown is responsible for inhibiting activity under physiological conditions. Using this preparation and commercial ultrafoil grids, we have over the past 4 months collected two datasets on a Titan Krios microscope. These datasets were used to produce two independent structures at 3.5 and 3.9 A resolution representing KdpFABC in the presence and absence of K+. For future studies, we will add ATP and PO4 ligands such as AMPPCP, ADP, AlF4, BeF3, VO4 in the presence and absence of K+ to this same preparation to stabilize additional intermediates in the reaction cycle and thus to evaluate their structural characteristics. In parallel, we will use biochemical assays to correlate these structures with the functional behavior of KdpFABC.
Project Details
Start Date
2019-06-15
End Date
2020-01-31
Status
Closed
Released Data Link
Team
Principal Investigator
Team Members
Related Publications
M. Sweet, H. Erdjument-Bromage, T. A. Neubert, D.L. Stokes. (2020) Action and Inaction of the Bacterial Potassium Pump KdpFABC. 2020 Biophysical Society Meeting Abstracts. Biophysical Journal, Supplement, Abstract.