Protein Docking and Steered Molecular Dynamics Reveal Alternative Regulatory Sites on the SERCA Calcium Transporter

Abstract

The transport activity of the calcium ATPase SERCA is modulated by an inhibitory interaction with a 52-residue transmembrane peptide, phospholamban (PLB). Biochemical and structural studies have revealed the primary inhibitory site on SERCA, but PLB has been hypothesized to interact with alternative sites on SERCA that are distinct from the inhibitory site. The present study was undertaken to test these hypotheses and explore structural determinants of SERCA regulation by PLB. Steered molecular dynamics (SMD) and membrane protein-protein docking experiments were performed to investigate the apparent affinity of PLB interactions with candidate sites on SERCA. We modeled the relative binding of PLB to several different conformations of SERCA, representing different enzymatic states sampled during the calcium transport catalytic cycle. Overall, the SMD and docking experiments suggest that the canonical binding site is preferred, but also provide evidence for alternative sites that are favorable for certain conformational states of SERCA.

Fluorescence lifetime imaging microscopy reveals sodium pump dimers in live cells

“…Docking simulations were performed using crystal structures of NKA that captured the E1 state of the pump (PDB: 3WGU ( 40 )). The complexes were obtained with Rosetta MPDock ( 41 , 42 ), generating 1000 configurations that were ranked based on the Rosetta interaction energy units. Inspection of the top ranked complexes yielded three configurations deemed to be plausible based on (1) correct topological orientation, (2) a substantial interaction surface, and (3) compatibility with estimates of FRET distances between α–α, α–PLM, and α−β labeling sites.…”

“…To explore the atomic details of the NKA dimer interface, we performed molecular docking of NKA dimers in different states of the reaction cycle using the following crystal structures: 3KDP (E2P) ( 60 ), 7DDJ (E2Pi) ( 61 ), and 3WGU (E1Pi.ADP) ( 40 ). Docking was performed following the strategy described in ( 41 ), in search of interactions between either homodimers or heterodimers (with 3KDP ( 60 ) in the E2P state). Specifically, we used ClusPro for global docking to generate possible starting structures.…”

Stoichiometry of the Sodium Pump-Phospholemman Regulatory Complex

“…Docking simulations were performed using crystal structures of NKA that captured the E1 state of the pump (PDB: 3WGU (Kanai et al, 2013)). The complexes were obtained with Rosetta MPDock (Alford et al, 2020(Alford et al, , 2015, generating 1000 configurations that were ranked based on the Rosetta interaction energy units. Inspection of the top ranked complexes yielded three configurations that satisfy both the correct orientation and a substantial surface of intramolecular interaction.…”

“…To explore the atomic details of the NKA dimer interface, we performed molecular docking of NKA dimers in different states of the reaction cycle using the following crystal structures: 3KDP (E2P) (Morth et al, 2007), 7DDJ (E2Pi) (Kanai et al, 2020), and 3WGU (E1Pi.ADP) (Kanai et al, 2013). Docking was performed following the strategy described in (Alford et al, 2020), in search of interactions between either homo-or heterodimers (with 3KDP (Morth et al, 2007) in the E2P state). Specifically, we used ClusPro for global docking to generate possible starting structures.…”