Phytochrome proteins control the growth, reproduction, and photosynthesis of plants, fungi, and bacteria. Light is detected by a bilin cofactor, but it remains elusive how this leads to activation of ...the protein through structural changes. We present serial femtosecond X-ray crystallographic data of the chromophore-binding domains of a bacterial phytochrome at delay times of 1 ps and 10 ps after photoexcitation. The data reveal a twist of the D-ring, which leads to partial detachment of the chromophore from the protein. Unexpectedly, the conserved so-called pyrrole water is photodissociated from the chromophore, concomitant with movement of the A-ring and a key signaling aspartate. The changes are wired together by ultrafast backbone and water movements around the chromophore, channeling them into signal transduction towards the output domains. We suggest that the observed collective changes are important for the phytochrome photoresponse, explaining the earliest steps of how plants, fungi and bacteria sense red light.
The crystal structure of the dimeric membrane domain of human Band 31, the red cell chloride/bicarbonate anion exchanger 1 (AE1, SLC4A1), provides a structural context for over four decades of ...studies into this historic and important membrane glycoprotein. In this review, we highlight the key structural features responsible for anion binding and translocation and have integrated the following topological markers within the Band 3 structure: blood group antigens, N-glycosylation site, protease cleavage sites, inhibitor and chemical labeling sites, and the results of scanning cysteine and N-glycosylation mutagenesis. Locations of mutations linked to human disease, including those responsible for Southeast Asian ovalocytosis, hereditary stomatocytosis, hereditary spherocytosis, and distal renal tubular acidosis, provide molecular insights into their effect on Band 3 folding. Finally, molecular dynamics simulations of phosphatidylcholine self-assembled around Band 3 provide a view of this membrane protein within a lipid bilayer.
Human Band 3 dimer in a lipid bilayer. Each subunit is colored using the rainbow color (blue to red) from N-terminal to C-terminal and the positions of the lipid phosphate groups shown as gray spheres. Display omitted
•The Band 3 chloride/bicarbonate anion exchanger 1 (AE1, SLC4A1) is one of the best-studied human membrane transport proteins.•A recent 3.5Å crystal structure of the Band 3 membrane domain provides new insights into its mechanism of action.•This review places over 40year of research on Band 3 into a structural context and explains why mutations lead to disease.•Molecular dynamics simulations produced the first molecular model of Band 3 in a lipid bilayer.
Overcoming the challenges of membrane protein crystallography Carpenter, Elisabeth P; Beis, Konstantinos; Cameron, Alexander D ...
Current opinion in structural biology,
October 2008, 2008-Oct, 2008-10-00, 20081001, Letnik:
18, Številka:
5
Journal Article
Recenzirano
Odprti dostop
Membrane protein structural biology is still a largely unconquered area, given that approximately 25% of all proteins are membrane proteins and yet less than 150 unique structures are available. ...Membrane proteins have proven to be difficult to study owing to their partially hydrophobic surfaces, flexibility and lack of stability. The field is now taking advantage of the high-throughput revolution in structural biology and methods are emerging for effective expression, solubilisation, purification and crystallisation of membrane proteins. These technical advances will lead to a rapid increase in the rate at which membrane protein structures are solved in the near future.
Abstract
In addition to the serotonin 5-HT
2A
receptor (5-HT
2A
R), the dopamine D
2
receptor (D
2
R) is a key therapeutic target of antipsychotics for the treatment of schizophrenia. The inactive ...state structures of D
2
R have been described in complex with the inverse agonists risperidone (D
2
R
ris
) and haloperidol (D
2
R
hal
). Here we describe the structure of human D
2
R in complex with spiperone (D
2
R
spi
). In D
2
R
spi
, the conformation of the extracellular loop (ECL) 2, which composes the ligand-binding pocket, was substantially different from those in D
2
R
ris
and D
2
R
hal
, demonstrating that ECL2 in D
2
R is highly dynamic. Moreover, D
2
R
spi
exhibited an extended binding pocket to accommodate spiperone’s phenyl ring, which probably contributes to the selectivity of spiperone to D
2
R and 5-HT
2A
R. Together with D
2
R
ris
and D
2
R
hal
, the structural information of D
2
R
spi
should be of value for designing novel antipsychotics with improved safety and efficacy.
Serial femtosecond X-ray crystallography (SFX) has revolutionized atomic-resolution structural investigation by expanding applicability to micrometer-sized protein crystals, even at room temperature, ...and by enabling dynamics studies. However, reliable crystal-carrying media for SFX are lacking. Here we introduce a grease-matrix carrier for protein microcrystals and obtain the structures of lysozyme, glucose isomerase, thaumatin and fatty acid-binding protein type 3 under ambient conditions at a resolution of or finer than 2 Å.
Enterobacteriaceae produce antimicrobial peptides for survival under nutrient starvation. Microcin J25 (MccJ25) is an antimicrobial peptide with a unique lasso topology. It is secreted by the ...ATP-binding cassette (ABC) exporter McjD, which ensures self-immunity of the producing strain through efficient export of the toxic mature peptide from the cell. Here we have determined the crystal structure of McjD from Escherichia coli at 2.7-Å resolution, which is to the authors’ knowledge the first structure of an antibacterial peptide ABC transporter. Our functional and biochemical analyses demonstrate McjD-dependent immunity to MccJ25 through efflux of the peptide. McjD can directly bind MccJ25 and displays a basal ATPase activity that is stimulated by MccJ25 in both detergent solution and proteoliposomes. McjD adopts a new conformation, termed nucleotide-bound outward occluded. The new conformation defines a clear cavity; mutagenesis and ligand binding studies of the cavity have identified Phe86, Asn134, and Asn302 as important for recognition of MccJ25. Comparisons with the inward-open MsbA and outward-open Sav1866 structures show that McjD has structural similarities with both states without the intertwining of transmembrane (TM) helices. The occluded state is formed by rotation of TMs 1 and 2 toward the equivalent TMs of the opposite monomer, unlike Sav1866 where they intertwine with TMs 3–6 of the opposite monomer. Cysteine cross-linking studies on the McjD dimer in inside-out membrane vesicles of E. coli confirmed the presence of the occluded state. We therefore propose that the outward-occluded state represents a transition intermediate between the outward-open and inward-open conformation of ABC exporters.
Nitric oxide reductase (NOR) is an iron-containing enzyme that catalyzes the reduction of nitric oxide (NO) to generate a major greenhouse gas, nitrous oxide (N₂O). Here, we report the crystal ...structure of NOR from Pseudomonas aeruginosa at 2.7 angstrom resolution. The structure reveals details of the catalytic binuclear center. The non-heme iron (FeB) is coordinated by three His and one Glu ligands, but a His-Tyr covalent linkage common in cytochrome oxidases (COX) is absent. This structural characteristic is crucial for NOR reaction. Although the overall structure of NOR is closely related to COX, neither the D- nor K-proton pathway, which connect the COX active center to the intracellular space, was observed. Protons required for the NOR reaction are probably provided from the extracellular side.
Short chain peptides are actively transported across membranes as an efficient route for dietary protein absorption and for maintaining cellular homeostasis. In mammals, peptide transport occurs via ...PepT1 and PepT2, which belong to the proton‐dependent oligopeptide transporter, or POT family. The recent crystal structure of a bacterial POT transporter confirmed that they belong to the major facilitator superfamily of secondary active transporters. Despite the functional characterization of POT family members in bacteria, fungi and mammals, a detailed model for peptide recognition and transport remains unavailable. In this study, we report the 3.3‐Å resolution crystal structure and functional characterization of a POT family transporter from the bacterium Streptococcus thermophilus. Crystallized in an inward open conformation the structure identifies a hinge‐like movement within the C‐terminal half of the transporter that facilitates opening of an intracellular gate controlling access to a central peptide‐binding site. Our associated functional data support a model for peptide transport that highlights the importance of salt bridge interactions in orchestrating alternating access within the POT family.
Proton‐dependent oligopeptide transporters are required for the uptake of diet‐derived peptides in all kingdoms of life. The crystal structure of a bacterial transporter in the inward open conformation, together with a published structure in an occluded conformation, reveals the peptide transport mechanism.
Orexin receptors are a family of G protein-coupled receptors that consist of two subtypes: orexin-1 receptors (OX1Rs) and OX2Rs. They are expressed throughout the central nervous system and are ...involved in regulating the sleep-wake cycle. The development of antagonists to orexin receptors has become important in drug discovery because modulation of these receptors can lead to novel treatments for diseases related to the regulation of sleep and wakefulness, such as insomnia. In this study, we determined that the structure of OX2R bound to lemborexant, a dual orexin receptor antagonist (DORA), at 2.89 Å resolution. Comparisons of kinetic and dynamic properties of DORAs based on structures and simulations suggest that the enthalpy of molecular binding to receptors and the entropy derived from intramolecular structure can be separately controlled. These results complement existing structural information and allow us to discuss the usefulness of pharmacophore models and target selectivity to OXRs.
Display omitted
•The structure of OX2R with the bound anti-insomnia drug lemborexant was determined•Binding mode of lemborexant differs between OXRs due to different Gln3.32 confomations•MD simulations show that OX2R preferentially binds lemborexant compared with suvorexant•Findings may facilitate the development of new anti-insomnia drugs
Asada et al. determined the crystal structure of orexin 2 receptor (OX2R) with the bound anti-insomnia drug lemborexant. The structure reveals important features of lemborexant binding to the receptor, which is of interest for further anti-insomnia drug development.
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