5 research outputs found
Initiating Heavy-atom Based Phasing by Multi-Dimensional Molecular Replacement
To obtain an electron-density map from a macromolecular crystal the
phase-problem needs to be solved, which often involves the use of heavy-atom
derivative crystals and concomitantly the determination of the heavy atom
substructure. This is customarily done by direct methods or Patterson-based
approaches, which however may fail when only poorly diffracting derivative
crystals are available, as often the case for e.g. membrane proteins. Here we
present an approach for heavy atom site identification based on a Molecular
Replacement Parameter Matrix (MRPM) search. It involves an n-dimensional search
to test a wide spectrum of molecular replacement parameters, such as clusters
of different conformations. The result is scored by the ability to identify
heavy-atom positions, from anomalous difference Fourier maps, that allow
meaningful phases to be determined. The strategy was successfully applied in
the determination of a membrane protein structure, the CopA Cu+-ATPase, when
other methods had failed to resolve the heavy atom substructure. MRPM is
particularly suited for proteins undergoing large conformational changes where
multiple search models should be generated, and it enables the identification
of weak but correct molecular replacement solutions with maximum contrast to
prime experimental phasing efforts.Comment: 19 pages total, main paper: 6 pages (2 figures), supplementary
material: 13 pages (2 figures, 9 tabels
Structural models of the human copper P-type ATPases ATP7A and ATP7B
The human copper exporters ATP7A and ATP7B contain domains common to all P-type ATPases as well as class-specific features such as six sequential heavy-metal binding domains (HMBD1-HMBD6) and a type-specific constellation of transmembrane helices. Despite the medical significance of ATP7A and ATP7B related to Menkes and Wilson diseases, respectively, structural information has only been available for isolated, soluble domains. Here we present homology models based on the existing structures of soluble domains and the recently determined structure of the homologous LpCopA from the bacterium Legionella pneumophila. The models and sequence analyses show that the domains and residues involved in the catalytic phosphorylation events and copper transfer are highly conserved. In addition, there are only minor differences in the core structures of the two human proteins and the bacterial template, allowing protein-specific properties to be addressed. Furthermore, the mapping of known disease-causing missense mutations indicates that among the heavy-metal binding domains, HMBD5 and HMBD6 are the most crucial for function, thus mimicking the single or dual HMBDs found in most copper-specific P-type ATPases. We propose a structural arrangement of the HMBDs and how they may interact with the core of the proteins to achieve autoinhibition
Structure and autoregulation of a P4-ATPase lipid flippase
International audienceType 4 P-type ATPases (P4-ATPases) are lipid flippases that drive the active transport of phospholipids from exoplasmic or luminal leaflets to cytosolic leaflets of eukaryotic membranes. The molecular architecture of P4-ATPases and the mechanism through which they recognize and transport lipids have remained unknown. Here we describe the cryo-electron microscopy structure of the P4-ATPase Drs2p-Cdc50p, a Saccharomyces cerevisiae lipid flippase that is specific to phosphatidylserine and phosphatidylethanolamine. Drs2p-Cdc50p is autoinhibited by the C-terminal tail of Drs2p, and activated by the lipid phosphatidylinositol-4-phosphate (PtdIns4P or PI4P). We present three structures that represent the complex in an autoinhibited, an intermediate and a fully activated state. The analysis highlights specific features of P4-ATPases and reveals sites of autoinhibition and PI4P-dependent activation. We also observe a putative lipid translocation pathway in this flippase that involves a conserved PISL motif in transmembrane segment 4 and polar residues of transmembrane segments 2 and 5, in particular Lys1018, in the centre of the lipid bilayer