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The global spread of multidrug-resistant

The global spread of multidrug-resistant Acinetobacter baumannii infections urgently calls for the identification of novel drug targets. We solved the electron cryo-microscopy structure of the F 1 F o –adenosine 5′-triphosphate (ATP) synthase from A. baumannii in three distinct conformational states. The nucleotide-converting F 1 subcomplex reveals a specific self-inhibition mechanism, which supports a unidirectional ratchet mechanism to avoid wasteful ATP consumption. In the membrane-embedded F o complex, the structure shows unique structural adaptations along both the entry and exit pathways of the proton-conducting a-subunit. These features, absent in mitochondrial ATP synthases, represent attractive targets for the development of next-generation therapeutics that can act directly at the culmination of bioenergetics in this clinically relevant pathogen.

Description, The structure of the hexameric fungal proton pump Pma1 reveals its mechanism of autoinhibition and interactions with lipids., The fungal plasma membrane H+-ATPase Pma1 is a vital enzyme, generating a proton-motive force that drives the import of essential nutrients. Autoinhibited Pma1 hexamers in the plasma membrane of starving fungi are activated by glucose signaling and subsequent phosphorylation of the autoinhibitory domain. As related P-type adenosine triphosphatases (ATPases) are not known to oligomerize, the physiological relevance of Pma1 hexamers remained unknown. We have determined the structure of hexameric Pma1 from Neurospora crassa by electron cryo-microscopy at 3.3-Å resolution, elucidating the molecular basis for hexamer formation and autoinhibition and providing a basis for structure-based drug development. Coarse-grained molecular dynamics simulations in a lipid bilayer suggest lipid-mediated contacts between monomers and a substantial protein-induced membrane deformation that could act as a proton-attracting funnel.

Acinetobacter baumannii is a clinically relevant pathogen which causes multi-drug resistant, hospital-acquired infections and is a top priority target for antibiotic development. Cryo-EM structures of the A. baumannii F1Fo-ATP synthase in three conformational states reveal unique features, which represent attractive sites for the development of novel therapeutics.One sentence summaryStructure of Acinetobacter baumannii ATP synthase

The fungal plasma membrane H+-ATPase Pma1 is a vital enzyme, generating a proton-motive force that drives the import of essential nutrients. Auto-inhibited Pma1 hexamers in starving fungi are activated by glucose signalling resulting in phosphorylation of the auto-inhibitory domain. As related P-type ATPases are not known to oligomerise, the physiological relevance of Pma1 hexamers remains unknown. We have determined the structure of hexameric Pma1 from Neurospora crassa by cryo-EM at 3.3 Å resolution, elucidating the molecular basis for hexamer formation and auto-inhibition, and providing a basis for structure-based drug development. Coarse-grained molecular dynamics simulations in a lipid bilayer suggest lipid-mediated contacts between monomers and a substantial protein-induced membrane deformation that could act as a proton-attracting funnel.

The discovery of the P-type ATPase sarcoplasmic/endoplasmic reticulum Ca2+-ATPase (SERCA) as a modulator of oncogenic NOTCH1 suggests an innovative approach for treating T-cell acute lymphoblastic leukemia (T-ALL). In fact, SERCA inhibition preferentially affects the maturation and activity of the most common class of oncogenic NOTCH1 mutants. The aim of this study was to identify inhibitors with better drug-like properties and reduced off-target toxicity for SERCA inhibition. We developed a novel oral SERCA inhibitor, CAD204520, through medicinal chemistry optimization and crystal structure-oriented analysis describing its anti-leukemic effects in vitro and in vivo to support a SERCA-based therapeutic modality in T-ALL. From a 191,000 small-molecule screening targeting P-type ATPase, we identified CAD204520 as a selective inhibitor of human SERCA compared to Na+/K+ and H+-ATPase. Crystal structure analysis showed that CAD204520 binds the transmembrane interface of SERCA between helices M1, M2, M3, and M4. CAD204520 minimally alters Ca2+ shift and fails to trigger Ca2+-dependent programs, such as the unfolded protein response. We demonstrated that CAD204520 impairs the proliferation of T-ALL cell lines carrying activating mutations of NOTCH1. Importantly, clinical samples carrying NOTCH1 mutations, including PEST deletions, were more sensitive to CAD204520 compared to normal lymphocytes or wild-type NOTCH1 ALL cells. Mechanistically, CAD204520 treatment reduces the levels of the activated form of NOTCH1 as a consequence of a defect in NOTCH1 trafficking. Next, we demonstrated that somatic hotspot mutations in the SERCA2 ATPase pocket that confer resistance to known SERCA modulators (e.g., thapsigargin) do not interfere with CAD204520 binding, suggesting that the activity of CAD204520 will be unlikely affected by recurrent resistant genetic variants. Finally, we showed that 30 mg/kg BID for 21 days is well-tolerated in vivo in CD1 mice without causing loss of weight and cardiac toxicity. In a xenograft SKW-3/KE-37 T-ALL model, CAD204520 reduces circulating and tissue-infiltrating human leukemia T-ALL cells with no heart-related or gastrointestinal toxicities. In conclusion, we present CAD204520 as a novel orally bioavailable SERCA inhibitor with tolerable off-target toxicity in NOTCH1-dependent tumors. This work provides a foundation for the further development of novel drugs targeting Notch-dependent hematopoietic malignancies.

Peptide interactions with lipid bilayers play a key role in a range of biological processes and depend on electrostatic interactions between charged amino acids and lipid headgroups. Antimicrobial peptides (AMPs) initiate the killing of bacteria by binding to and destabilizing their membranes. The multiple peptide resistance factor (MprF) provides a defense mechanism for bacteria against a broad range of AMPs. MprF reduces the negative charge of bacterial membranes through enzymatic conversion of the anionic lipid phosphatidyl glycerol (PG) to either zwitterionic alanyl-phosphatidyl glycerol (Ala-PG) or cationic lysyl-phosphatidyl glycerol (Lys-PG). The resulting change in the membrane charge is suggested to reduce the binding of AMPs to membranes, thus impeding downstream AMP activity. Using coarse-grained molecular dynamics to investigate the effects of these modified lipids on AMP binding to model membranes, we show that AMPs have substantially reduced affinity for model membranes containing Ala-PG or Lys-PG. More than 5000 simulations in total are used to define the relationship between lipid bilayer composition, peptide sequence (using five different membrane-active peptides), and peptide binding to membranes. The degree of interaction of a peptide with a membrane correlates with the membrane surface charge density. Free energy profile (potential of mean force) calculations reveal that the lipid modifications due to MprF alter the energy barrier to peptide helix penetration of the bilayer. These results will offer a guide to the design of novel peptides, which addresses the issue of resistance via MprF-mediated membrane modification.

International audience; The sarco(endo)plasmic reticulum Ca 2+ -ATPase (SERCA) is a P-type ATPase that transports Ca 2+ from the cytosol into the sarco(endo)plasmic reticulum (SR/ER) lumen, driven by ATP. This primary transport activity depends on tight coupling between movements of the transmembrane helices forming the two Ca 2+ -binding sites and the cytosolic headpiece mediating ATP hydrolysis. We have addressed the molecular basis for this intramolecular communication by analyzing the structure and functional properties of the SERCA mutant E340A. The mutated Glu340 residue is strictly conserved among the P-type ATPase family of membrane transporters and is located at a seemingly strategic position at the interface between the phosphorylation domain and the cytosolic ends of 5 of SERCA’s 10 transmembrane helices. The mutant displays a marked slowing of the Ca 2+ -binding kinetics, and its crystal structure in the presence of Ca 2+ and ATP analog reveals a rotated headpiece, altered connectivity between the cytosolic domains, and an altered hydrogen bonding pattern around residue 340. Supported by molecular dynamics simulations, we conclude that the E340A mutation causes a stabilization of the Ca 2+ sites in a more occluded state, hence displaying slowed dynamics. This finding underpins a crucial role of Glu340 in interdomain communication between the headpiece and the Ca 2+ -binding transmembrane region.

Significance We present a crystal structure, functional data, and molecular dynamics (MD) simulations of the sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) mutant E340A. The mutation slows Ca2+-binding kinetics, and the structural differences between wild type and E340A indicate that the mutation disrupts a central interdomain “communication hub” governing Ca2+ binding/dissociation. MD simulations reveal altered dynamics in regions mediating Ca2+ occlusion, a critical step in SERCA’s alternating access mechanism. The mutation stabilizes a more occluded state of the Ca2+ sites. The strict conservation of Glu340 among P-type ATPases is the result of its critical role in interdomain communication between the cytosolic headpiece and the transmembrane domain, ensuring a delicate balance between dynamics of ion binding, occlusion, and release—key steps in the transport process., The sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) is a P-type ATPase that transports Ca2+ from the cytosol into the sarco(endo)plasmic reticulum (SR/ER) lumen, driven by ATP. This primary transport activity depends on tight coupling between movements of the transmembrane helices forming the two Ca2+-binding sites and the cytosolic headpiece mediating ATP hydrolysis. We have addressed the molecular basis for this intramolecular communication by analyzing the structure and functional properties of the SERCA mutant E340A. The mutated Glu340 residue is strictly conserved among the P-type ATPase family of membrane transporters and is located at a seemingly strategic position at the interface between the phosphorylation domain and the cytosolic ends of 5 of SERCA’s 10 transmembrane helices. The mutant displays a marked slowing of the Ca2+-binding kinetics, and its crystal structure in the presence of Ca2+ and ATP analog reveals a rotated headpiece, altered connectivity between the cytosolic domains, and an altered hydrogen bonding pattern around residue 340. Supported by molecular dynamics simulations, we conclude that the E340A mutation causes a stabilization of the Ca2+ sites in a more occluded state, hence displaying slowed dynamics. This finding underpins a crucial role of Glu340 in interdomain communication between the headpiece and the Ca2+-binding transmembrane region.

The sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) is a P-type ATPase that transports Ca2+from the cytosol into the SR/ER lumen, driven by ATP. This primary transport activity depends on tight coupling between movements of the transmembrane helices forming the two Ca2+binding sites and of the cytosolic headpiece mediating ATP hydrolysis. We have addressed the molecular basis for this intramolecular communication by analyzing the structure and functional properties of the SERCA mutant E340A. The mutated Glu340 residue is strictly conserved among the P-type ATPase family of membrane transporters and is located at a seemingly strategic position at the interface between the phosphorylation domain and the cytosolic ends of five out of SERCA’s ten transmembrane helices. The mutant displays a marked slowing of the Ca2+-binding kinetics, and its crystal structure in the presence of Ca2+and ATP analogue reveals a rotated headpiece, altered connectivity between the cytosolic domains and altered hydrogen bonding pattern around residue 340. Supported by molecular dynamics simulations, we conclude that the E340A mutation causes a stabilization of the Ca2+sites in a more occluded state, hence displaying slowed dynamics. This finding underpins a crucial role of Glu340 in inter-domain communication between the headpiece and the Ca2+-binding transmembrane region.

The identification of SERCA (sarco/endoplasmic reticulum calcium ATPase) as a target for modulating gain-of-function NOTCH1 mutations in Notch-dependent cancers has spurred the development of this compound class for cancer therapeutics. Despite the innate toxicity challenge associated with SERCA inhibition, we identified CAD204520, a small molecule with better drug-like properties and reduced off-target Ca2+ toxicity compared with the SERCA inhibitor thapsigargin. In this work, we describe the properties and complex structure of CAD204520 and show that CAD204520 preferentially targets mutated over wild-type NOTCH1 proteins in T cell acute lymphoblastic leukemia (T-ALL) and mantle cell lymphoma (MCL). Uniquely among SERCA inhibitors, CAD204520 suppresses NOTCH1-mutated leukemic cells in a T-ALL xenografted model without causing cardiac toxicity. This study supports the development of SERCA inhibitors for Notch-dependent cancers and extends their application to cases with isolated mutations in the PEST degradation domain of NOTCH1, such as MCL or chronic lymphocytic leukemia (CLL). Clinical translation of SERCA inhibitors has been hampered by the risk of adverse cardiac events. In this work, Marchesini and Gherli et al. identified a tolerable oral available SERCA inhibitor, CAD204520, and showed that modulation of clinically relevant NOTCH1 mutations in T cell acute lymphoblastic leukemia and mantle cell lymphoma.

Antimicrobial peptides (AMPs) initiate killing of bacteria by binding to and destabilizing their membranes. The multiple peptide resistance factor (MprF) provides a defence mechanism for bacteria against a broad range of AMPs. MprF reduces the negative charge of both Gram-positive and Gram--negative bacterial membranes through enzymatic conversion of the anionic lipid phosphatidyl glycerol (PG) to either zwitterionic alanyl-phosphatidyl glycerol (Ala-PG) or cationic lysylphosphatidyl glycerol (Lys-PG). The resulting change in membrane charge is suggested to reduce AMP-membrane binding and hinder downstream AMP activity. Using molecular dynamics to investigate the effects of these modified lipids on AMP-binding to model membranes, we show that AMPs have substantially reduced affinity for model membranes containing Ala-PG or Lys-PG. A total of ~7000 simulations are used to define the relationship between bilayer composition and binding for 5 different membrane active peptides. The reduction of degree of interaction of a peptide with the membrane is shown to correlate with the change in membrane surface charge density. Free energy profile (potential of mean force) calculations reveal that these lipid modifications alter the energy barrier to peptide helix penetration of the bilayer. These results will enable us to guide design of novel peptides which address the issue of resistance via MprF-mediated membrane modification.

This month’s Under the Lens discusses recent advances in high-resolution imaging of bacterial membrane proteins within intact liposomes by electron cryomicroscopy.

The activity of the sarco/endoplasmic reticulum Ca(2+) -ATPase (SERCA) depends strongly on the lipid composition of the surrounding membrane. Yet, structural information on SERCA-lipid interaction is still relatively scarce, and the influence of different lipids on the enzyme is not well understood. We have analyzed SERCA crystal structures in the presence of four different phosphatidylcholine lipids of different lengths and double-bond compositions, and we find three different binding sites for lipid head groups, which are apparently independent of the acyl moiety of the lipids used. By comparison with other available SERCA structures with bound lipids, we find a total of five recurring sites, two of which are specific to certain conformational states of the enzyme, two others are state-independent, and one is a crucial site for crystal formation. Three of the binding sites overlap with or are in close vicinity to known binding sites for various SERCA-specific inhibitors and regulators, e.g. thapsigargin, sarcolipin/phospholamban and cyclopiazonic acid. Whereas the transient sites are amenable to a transient, regulatory influence of lipid molecules, the state-independent sites probably provide a flexible anchoring of the protein in the fluid bilayer.

The sarco(endo)plasmic reticulum Ca 2+ ‐ATPase (SERCA) couples ATP hydrolysis to transport of Ca 2+ . This directed energy transfer requires cross‐talk between the two Ca 2+ sites and the phosphorylation site over 50 A distance. We have addressed the mechano‐structural basis for this intramolecular signal by analysing the structure and the functional properties of SERCA mutant E309Q. Glu 309 contributes to Ca 2+ coordination at site II, and a consensus has been that E309Q only binds Ca 2+ at site I. The crystal structure of E309Q in the presence of Ca 2+ and an ATP analogue, however, reveals two occupied Ca 2+ sites of a non‐catalytic Ca 2 E 1 state. Ca 2+ is bound with micromolar affinity by both Ca 2+ sites in E309Q, but without cooperativity. The Ca 2+ ‐bound mutant does phosphorylate from ATP, but at a very low maximal rate. Phosphorylation depends on the correct positioning of the A‐domain, requiring a shift of transmembrane segment M1 into an ‘up and kinked position’. This transition is impaired in the E309Q mutant, most likely due to a lack of charge neutralization and altered hydrogen binding capacities at Ca 2+ site II.

The contraction and relaxation of muscle cells is controlled by the successive rise and fall of cytosolic Ca(2+), initiated by the release of Ca(2+) from the sarcoplasmic reticulum and terminated by re-sequestration of Ca(2+) into the sarcoplasmic reticulum as the main mechanism of Ca(2+) removal. Re-sequestration requires active transport and is catalysed by the sarcoplasmic reticulum Ca(2+)-ATPase (SERCA), which has a key role in defining the contractile properties of skeletal and heart muscle tissue. The activity of SERCA is regulated by two small, homologous membrane proteins called phospholamban (PLB, also known as PLN) and sarcolipin (SLN). Detailed structural information explaining this regulatory mechanism has been lacking, and the structural features defining the pathway through which cytoplasmic Ca(2+) enters the intramembranous binding sites of SERCA have remained unknown. Here we report the crystal structure of rabbit SERCA1a (also known as ATP2A1) in complex with SLN at 3.1 Å resolution. The regulatory SLN traps the Ca(2+)-ATPase in a previously undescribed E1 state, with exposure of the Ca(2+) sites through an open cytoplasmic pathway stabilized by Mg(2+). The structure suggests a mechanism for selective Ca(2+) loading and activation of SERCA, and provides new insight into how SLN and PLB inhibition arises from stabilization of this E1 intermediate state without bound Ca(2+). These findings may prove useful in studying how autoinhibitory domains of other ion pumps modulate transport across biological membranes.

Vanadate is the hallmark inhibitor of the P-type ATPase family; however, structural details of its inhibitory mechanism have remained unresolved. We have determined the crystal structure of sarcoplasmic reticulum Ca(2+)-ATPase with bound vanadate in the absence of Ca(2+). Vanadate is bound at the catalytic site as a planar VO3(-) in complex with water and Mg(2+) in a dephosphorylation transition-state-like conformation. Validating bound VO3(-) by anomalous difference Fourier maps using long-wavelength data we also identify a hitherto undescribed Cl(-) site near the dephosphorylation site. Crystallization was facilitated by trinitrophenyl (TNP)-derivatized nucleotides that bind with the TNP moiety occupying the binding pocket that normally accommodates the adenine of ATP, rationalizing their remarkably high affinity for E2P-like conformations of the Ca(2+)-ATPase. A comparison of the configurations of bound nucleotide analogs in the E2·VO3(-) structure with that in E2·BeF3(-) (E2P ground state analog) reveals multiple binding modes to the Ca(2+)-ATPase.

The Na+/K+-ATPase enzyme pumps three sodium ions out of the cell and two potassium ions into the cell while splitting a single molecule of ATP. This process generates the chemical gradients across the plasma membrane that are essential to electrical excitation and ion balance in animal cells. Here the authors determine that the C-terminus of the ATPase is a key regulator of a previously unrecognized ion pathway. Their data suggest that a cytoplasmic proton can enter and stabilize site III when empty (in the potassium-bound state); when the potassium ion is released, the proton returns to the cytoplasm, thus permitting an overall asymmetric stoichiometry of the transported ions. The C-terminus is crucial for pump function as demonstrated by reports that at least eight mutations in the region cause severe neurological diseases. The Na+/K+-ATPase pumps three sodium ions out of and two potassium ions into the cell while splitting a single molecule of ATP. Here it is found that the carboxy terminus of the ATPase's α-subunit is also a key regulator of a previously unrecognized ion pathway. The data indicate that, in the ATPase's potassium-bound state, a cytoplasmic proton can enter and stabilize site III when empty. When potassium is released, the proton returns to the cytoplasm, thus permitting an overall asymmetric stoichiometry of the transported ions. The Na+/K+-ATPase pumps three sodium ions out of and two potassium ions into the cell for each ATP molecule that is split, thereby generating the chemical and electrical gradients across the plasma membrane that are essential in, for example, signalling, secondary transport and volume regulation in animal cells. Crystal structures of the potassium-bound form of the pump revealed an intimate docking of the α-subunit carboxy terminus at the transmembrane domain1,2. Here we show that this element is a key regulator of a previously unrecognized ion pathway. Current models of P-type ATPases operate with a single ion conduit through the pump3,4,5, but our data suggest an additional pathway in the Na+/K+-ATPase between the ion-binding sites and the cytoplasm. The C-terminal pathway allows a cytoplasmic proton to enter and stabilize site III when empty in the potassium-bound state, and when potassium is released the proton will also return to the cytoplasm, thus allowing an overall asymmetric stoichiometry of the transported ions. The C terminus controls the gate to the pathway. Its structure is crucial for pump function, as demonstrated by at least eight mutations in the region that cause severe neurological diseases6,7. This novel model for ion transport by the Na+/K+-ATPase is established by electrophysiological studies of C-terminal mutations in familial hemiplegic migraine 2 (FHM2) and is further substantiated by molecular dynamics simulations. A similar ion regulation is likely to apply to the H+/K+-ATPase and the Ca2+-ATPase.

We have determined the structure of the sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) in an E2·Pi-like form stabilized as a complex with \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}{\mathrm{MgF}}_{4}^{2-}\end{equation*}\end{document}, an ATP analog, adenosine 5′-(β,γ-methylene)triphosphate (AMPPCP), and cyclopiazonic acid (CPA). The structure determined at 2.5Å resolution leads to a significantly revised model of CPA binding when compared with earlier reports. It shows that a divalent metal ion is required for CPA binding through coordination of the tetramic acid moiety at a characteristic kink of the M1 helix found in all P-type ATPase structures, which is expected to be part of the cytoplasmic cation access pathway. Our model is consistent with the biochemical data on CPA function and provides new measures in structure-based drug design targeting Ca2+-ATPases, e.g. from pathogens. We also present an extended structural basis of ATP modulation pinpointing key residues at or near the ATP binding site. A structural comparison to the Na+,K+-ATPase reveals that the Phe93 side chain occupies the equivalent binding pocket of the CPA site in SERCA, suggesting an important role of this residue in stabilization of the potassium-occluded E2 state of Na+,K+-ATPase.

In the past 15 years, a large body of structural information on P-type ATPases has accumulated in the Protein Data Bank. The available crystal structures cover different enzymes in a variety of conformational states that are associated with the enzymatic activity of ATP-dependent ion translocation across membranes. This chapter provides an overview about the available structural information, along with some practical instructions on how to make meaningful comparisons of structures in different conformations, and how to generate morphs between series of structures, in order to analyze domain movements and structural flexibility.

In the past 15 years, a large body of structural information on P-type ATPases has accumulated in the Protein Data Bank. The available crystal structures cover different enzymes in a variety of conformational states that are associated with the enzymatic activity of ATP-dependent ion translocation across membranes. This chapter provides an overview about the available structural information, along with some practical instructions on how to make meaningful comparisons of structures in different conformations, and how to generate morphs between series of structures, in order to analyze domain movements and structural flexibility.

Proteins fold into three-dimensional structures, which determine their diverse functions. The conformation of the backbone of each structure is locally at each Cα effectively described by conformational angles resulting in Ramachandran plots. These, however, do not describe the conformations around hydrogen bonds, which can be non-local along the backbone and are of major importance for protein structure. Here, we introduce the spatial rotation between hydrogen bonded peptide planes as a new descriptor for protein structure locally around a hydrogen bond. Strikingly, this rotational descriptor sampled over high-quality structures from the protein data base (PDB) concentrates into 30 localized clusters, some of which correlate to the common secondary structures and others to more special motifs, yet generally providing a unifying systematic classification of local structure around protein hydrogen bonds. It further provides a uniform vocabulary for comparison of protein structure near hydrogen bonds even between bonds in different proteins without alignment.

International audience; Sarcolipin (SLN) is a regulatory peptide present in sarcoplasmic reticulum (SR) from skeletal muscle of animals. We find that native rabbit SLN is modified by a fatty acid anchor on Cys-9 with a palmitic acid in about 60% and, surprisingly, an oleic acid in the remaining 40%. SLN used for co-crystallization with SERCA1a (Winther, A. M., Bublitz, M., Karlsen, J. L., Moller, J. V., Hansen, J. B., Nissen, P., and Buch-Pedersen, M. J. (2013) Nature 495, 265-2691; Ref. 1) is also palmitoylated/oleoylated, but is not visible in crystal structures, probably due to disorder. Treatment with 1 m hydroxylamine for 1 h removes the fatty acids from a majority of the SLN pool. This treatment did not modify the SERCA1a affinity for Ca(2+) but increased the Ca(2+)-dependent ATPase activity of SR membranes indicating that the S-acylation of SLN or of other proteins is required for this effect on SERCA1a. Pig SLN is also fully palmitoylated/oleoylated on its Cys-9 residue, but in a reverse ratio of about 40/60. An alignment of 67 SLN sequences from the protein databases shows that 19 of them contain a cysteine and the rest a phenylalanine at position 9. Based on a cladogram, we postulate that the mutation from phenylalanine to cysteine in some species is the result of an evolutionary convergence. We suggest that, besides phosphorylation, S-acylation/deacylation also regulates SLN activity.

Proteins that contain metal cofactors are expected to be highly radiation sensitive since the degree of X-ray absorption correlates with the presence of high-atomic-number elements and X-ray energy. To explore the effects of local damage in serial femtosecond crystallography (SFX),Clostridium ferredoxinwas used as a model system. The protein contains two [4Fe–4S] clusters that serve as sensitive probes for radiation-induced electronic and structural changes. High-dose room-temperature SFX datasets were collected at the Linac Coherent Light Source of ferredoxin microcrystals. Difference electron density maps calculated from high-dose SFX and synchrotron data show peaks at the iron positions of the clusters, indicative of decrease of atomic scattering factors due to ionization. The electron density of the two [4Fe–4S] clusters differs in the FEL data, but not in the synchrotron data. Since the clusters differ in their detailed architecture, this observation is suggestive of an influence of the molecular bonding and geometry on the atomic displacement dynamics following initial photoionization. The experiments are complemented by plasma code calculations.

Proteins fold into three-dimensional structures, which determine their diverse functions. The conformation of the backbone of each structure is locally at each C(α) effectively described by conformational angles resulting in Ramachandran plots. These, however, do not describe the conformations around hydrogen bonds, which can be non-local along the backbone and are of major importance for protein structure. Here, we introduce the spatial rotation between hydrogen bonded peptide planes as a new descriptor for protein structure locally around a hydrogen bond. Strikingly, this rotational descriptor sampled over high-quality structures from the protein data base (PDB) concentrates into 30 localized clusters, some of which correlate to the common secondary structures and others to more special motifs, yet generally providing a unifying systematic classification of local structure around protein hydrogen bonds. It further provides a uniform vocabulary for comparison of protein structure near hydrogen bonds even between bonds in different proteins without alignment.

A crystal structure suggests four water molecules are present in the binding cavity of thapsigargin in sarco/endoplasmic reticulum calcium ATPase (SERCA). Computational chemistry indicates that three of these water molecules mediate an extensive hydrogen-bonding network between thapsigargin and the backbone of SERCA. The orientation of the thapsigargin molecule in SERCA is crucially dependent on these interactions. The hypothesis has been verified by measuring the affinity of newly synthesized model compounds, which are prevented from participating in such water-mediated interactions as hydrogen-bond donors.

The sarco/endoplasmic reticulum Ca(2+)-ATPase (SERCA) is a transmembrane ion transporter belonging to the P(II)-type ATPase family. It performs the vital task of re-sequestering cytoplasmic Ca(2+) to the sarco/endoplasmic reticulum store, thereby also terminating Ca(2+)-induced signaling such as in muscle contraction. This minireview focuses on the transport pathways of Ca(2+) and H(+) ions across the lipid bilayer through SERCA. The ion-binding sites of SERCA are accessible from either the cytoplasm or the sarco/endoplasmic reticulum lumen, and the Ca(2+) entry and exit channels are both formed mainly by rearrangements of four N-terminal transmembrane α-helices. Recent improvements in the resolution of the crystal structures of rabbit SERCA1a have revealed a hydrated pathway in the C-terminal transmembrane region leading from the ion-binding sites to the cytosol. A comparison of different SERCA conformations reveals that this C-terminal pathway is exclusive to Ca(2+)-free E2 states, suggesting that it may play a functional role in proton release from the ion-binding sites. This is in agreement with molecular dynamics simulations and mutational studies and is in striking analogy to a similar pathway recently described for the related sodium pump. We therefore suggest a model for the ion exchange mechanism in P(II)-ATPases including not one, but two cytoplasmic pathways working in concert.

P-type ATPases are a large family of integral membrane transporters that are of vital importance in all kingdoms of life. In eponymous distinction from the other main classes of transport ATPase – the FF1 (F-), the vacuolar (V-) and the ATP-binding cassette (ABC-) type – the P-type ATPases form

To obtain the initial crystallization hit and the subsequent optimization remain the major bottlenecks in membrane protein structure determination. Here we describe an approach to achieve diffraction quality membrane protein crystals in high concentrations of lipid and detergent (HILIDE). Initial hits are achieved through a controlled variation of the membrane protein/lipid/detergent ratios, and optimized crystals are established by systematic screening with secondary detergents at concentrations that are adapted for the method. The approach is generally applicable for native and heterologously expressed membrane proteins, and it is compatible with any crystallization method, such as vapor diffusion and batch crystallization under oil. The method provides a user defined lipidic environment and is in itself not limited to lipids with particular properties, such as the lipid-phase crystallization methods.

We have determined the structure of the sarco(endo) plasmic reticulum Ca2+-ATPase (SERCA) in an E2.P-i-like form stabilized as a complex with MgF42-, an ATP analog, adenosine 5'-(beta,gamma-methylene) triphosphate (AMPPCP), and cyclopiazonic acid (CPA). The structure determined at 2.5 angstrom resolution leads to a significantly revised model of CPA binding when compared with earlier reports. It shows that a divalent metal ion is required for CPA binding through coordination of the tetramic acid moiety at a characteristic kink of the M1 helix found in all P-type ATPase structures, which is expected to be part of the cytoplasmic cation access pathway. Our model is consistent with the biochemical data on CPA function and provides new measures in structure-based drug design targeting Ca2+-ATPases, e. g. from pathogens. We also present an extended structural basis of ATP modulation pinpointing key residues at or near the ATP binding site. A structural comparison to the Na+, K+-ATPase reveals that the Phe(93) side chain occupies the equivalent binding pocket of the CPA site in SERCA, suggesting an important role of this residue in stabilization of the potassium-occluded E2 state of Na+, K+-ATPase.

During a bacterial infection, each successive step is orchestrated by a dedicated set of virulence factors. In Gram-positive bacteria, the presentation or release of such factors is crucially dependent on the continual remodelling of the cell wall. We have investigated the autolysin or peptidoglycan hydrolase Auto (Lmo1076) from the human pathogen Listeria monocytogenes to structurally and biochemically underpin its role in host cell invasion. We demonstrate that Auto is an N-acetylglucosaminidase, that it is autoinhibited when newly secreted but activated by proteolytic cleavage, that it has an acidic pH optimum and that it preferentially cleaves acetylated over de-acetylated peptidoglycan. The crystal structure of Auto, the first for glycoside hydrolase family 73, and the first for a listerial autolysin, indicates that autoinhibition is due to an N-terminal alpha-helix unique to Auto that physically blocks the substrate-binding cleft. We identify Glu122 and Glu156 as the two catalytically essential carboxylate groups. The physical properties of Auto as well as its localization to lipoteichoic acid by its four C-terminal GW modules imply cell wall degradation by Auto to be highly co-ordinated. Its spatio-temporally controlled activation and localized activity in an acidified environment indicate that it facilitates remodelling of the cell wall and may be involved in co-ordinating the release of virulence factors at specific stages of an infection.

International audience; We report on the crystal structure of the internalin domain of InlJ, a virulence-associated surface protein of Listeria monocytogenes, at 2.7-Å resolution. InlJ is a member of the internalin family of listerial cell surface proteins characterized by a common N-terminal domain. InlJ bears 15 leucine-rich repeats (LRRs), the same number as in InlA, the prototypical internalin family member. The LRRs of InlJ differ from those of other internalins by having 21, rather than 22, residues and by replacing 1 LRR defining hydrophobic residue with a conserved cysteine. These cysteines stack to form an intramolecular ladder and regular hydrophobic interactions in consecutive repeats. Analyzing the curvature, twist, and lateral bending angles of InlJ and comparing these with several other LRR proteins, we provide a systematic geometric comparison of LRR protein structures (http://bragi2.helmholtz-hzi.de/Angulator/). These indicate that both cysteine and asparagine ladders stabilize the LRR fold, whereas substitutions in some repeat positions are more likely than others to induce changes in LRR geometry.

International audience