PELDOR Spectroscopy Reveals Two Defined States of a Sialic Acid TRAP Transporter SBP in Solution

Abstract

The tripartite ATP-independent periplasmic (TRAP) transporters are a widespread class of membrane transporters in bacteria and archaea. Typical substrates for TRAP transporters are organic acids including the sialic acid N-acetylneuraminic acid. The substrate binding proteins (SBP) of TRAP transporters are the best studied component and are responsible for initial high-affinity substrate binding. To better understand the dynamics of the ligand binding process, pulsed electron-electron double resonance (PELDOR, also known as DEER) spectroscopy was applied to study the conformational changes in the N-acetylneuraminic acid-specific SBP VcSiaP. The protein is the SBP of VcSiaPQM, a sialic acid TRAP transporter from Vibrio cholerae. Spin-labeled double-cysteine mutants of VcSiaP were analyzed in the substrate-bound and -free state and the measured distances were compared to available crystal structures. The data were compatible with two clear states only, which are consistent with the open and closed forms seen in TRAP SBP crystal structures. Substrate titration experiments demonstrated the transition of the population from one state to the other with no other observed forms. Mutants of key residues involved in ligand binding and/or proposed to be involved in domain closure were produced and the corresponding PELDOR experiments reveal important insights into the open-closed transition. The results are in excellent agreement with previous in vivo sialylation experiments. The structure of the spin-labeled Q54R1/L173R1 R125A mutant was solved at 2.1 Å resolution, revealing no significant changes in the protein structure. Thus, the loss of domain closure appears to be solely due to loss of binding. In conclusion, these data are consistent with TRAP SBPs undergoing a simple two-state transition from an openunliganded to closed-liganded state during the transport cycle.

Altered conformational sampling along an evolutionary trajectory changes the catalytic activity of an enzyme

“…Although the open conformation is the ground state for most ligand-free SBPs 1,5,[8][9][10][11][12][13][14][15][16][17][18][19] , numerous SBPs also sample semi-closed and closed states in the absence of ligands. For example, X-ray crystallography, nuclear magnetic resonance (NMR), molecular dynamics (MD) simulation, double electron-electron resonance (DEER, a.k.a.…”

“…PELDOR) and Förster resonance energy transfer (FRET) studies on the maltose-binding protein (MBP) [20][21][22] , glucose-galactose-binding protein 23,24 , histidine-binding protein 25 , ferri-bacillibactin-binding protein 26 , glutamine binding protein 27,28 and choline/acetylcholine binding protein 29 fundamental to determining the binding affinity 21,23,30,31 and binding promiscuity 5,32 of SBPs, and controlling the transport activity of SBP-associated systems 5,28 . Indeed, the extent of the open/closed motion differs between SBPs 1 , and the function of an SBP can be changed by mutations that alter conformational sampling, without changing the architecture of the SBP-ligand interface 19,21,30 .…”

The contribution of modern EPR to structural biology

“…Based on the Anfinsen dogma, it became customary to explain function in terms of a single conformation or of well-defined transitions between a few conformations defined at atomic resolution. While this is certainly a reasonable approximation in some cases [75][76][77], availability of distance distributions demonstrates that rather often conformation transitions are coupled to order-disorder transitions or are shifts in disorder equilibria [39,[78][79][80][81][82][83][84][85][86][87][88][89][90][91][92][93][94]. Among the systems addressed by PDS to date, the fraction where at least one state is genuinely disordered is surprisingly large.…”

Rates and equilibrium constants of the ligand-induced conformational transition of an HCN ion channel protein domain determined by DEER spectroscopy

“…This was the case encountered in a recent study where substrate binding to the substrate binding domain of a transporter was determined by DEER. 7 However, given our values of K A and K B , this limit is not applicable to cAMP binding to CNBD, and we conclude that the four-state model is necessary. The same four-state model with three equilibria has also been found optimal in a recent single-molecule FRET study of the CNBD-cAMP binding 27 .…”

“…6 DEER can also be used as a quantitative method for determining equilibrium constants such as p K a values 3 and substrate dissociation constants. 7 In principle, the time course of conformational changes of a protein induced by substrate or ligand binding can be probed by combining rapid freeze quench (RFQ) and EPR distance measurements.…”