What Can Be Learned About the Function of a Single Protein from Its Various X-Ray Structures: The Example of the Sarcoplasmic Calcium Pump

Abstract

Improvements in the handling of membrane proteins for crystallization, combined with better synchrotron sources for X-ray diffraction analysis, are leading to clarification of the structural details of an ever increasing number of membrane transporters and receptors. Here we describe how this development has resulted in the elucidation at atomic resolution of a large number of structures of the sarcoplasmic Ca(2+)-ATPase (SERCA1a) present in skeletal muscle. The structures corresponding to the various intermediary states have been obtained after stabilization with structural analogues of ATP and of metal fluorides as mimicks of inorganic phosphate. From these results it is possible, in accordance with previous biochemical and molecular biology data, to give a detailed structural description of both ATP hydrolysis and Ca(2+) transport through the membrane, to serve as the starting point for a fuller understanding of the pump mechanism and, in future studies, on the regulatory role of this ubiquitous intracellular Ca(2+)-ATPase in cellular Ca(2+) metabolism in normal and pathological conditions.

New Structures and Gating of Voltage-Dependent Potassium (Kv) Channels and Their Relatives: A Multi-Domain and Dynamic Question

“…Nevertheless, the possibility of using cryo-EM as a tool to capture different conformational states of the same protein, in proportion to their steady-state distribution [8], or in the presence of different pharmacological probes [88], increases the interest on this methodology. This could be reminiscent of the great contribution to understand the working mechanism of P-type ATPases, provided by elucidation of the crystal structures of Ca 2+ -ATPase in different conformational states [154,155,156,157,158,159]. Some remarkable steps in this direction are: (i) the recent comparison of SK channel activated and deactivated states, through three-dimensional classification of cryo-EM particles, to gain insights about the structural basis for channel activation [55], and (ii) the structural titration of the Na + -activated K + channel Slo2.2 with increasing concentrations of Na + , to detect an ensemble of closed conformations that do not depend on Na + concentration, followed by the emergence of an open conformation in a highly Na + -dependent way, without evidence of Na + -dependent intermediates [113,160].…”

Tracing Cytoplasmic Ca2+ Ion and Water Access Points in the Ca2+-ATPase

“…SERCA is one of the best structurally characterized membrane transporters. The first structure was determined in 2000 (6) and since then, numerous high-resolution structures have become available for several different functional states (7)(8)(9), revealing key details about the transport mechanism of the protein, as recently reviewed by Møller et al (10). The protein is composed of three cytoplasmic domains and a transmembrane (TM) domain consisting of 10 helices, here referred to as TM1-10 ( Fig.…”

Folding and stability of integral membrane proteins in amphipols

“…1A) is a poly(acrylic acid) 147 partially amidated with octylamine and isopropylamine, leaving 148 $35% of the carboxylic groups free [46]. In aqueous solution at 149 pH > 7, A8-35 assembles into small micelle-like particles with a 150 mass of $40 kDa [74][75][76]. Other APols developed more recently 151 include glycosylated nonionic APols (NAPols) [77,78] [50,51,73]).…”