Infection of the human stomach by Helicobacter pylori remains a worldwide problem and greatly contributes to peptic ulcer disease and gastric cancer. Without active intervention approximately 50% of ...the world population will continue to be infected with this gastric pathogen. Current eradication, called triple therapy, entails a proton-pump inhibitor and two broadband antibiotics, however resistance to either clarithromycin or metronidazole is greater than 25% and rising. Therefore, there is an urgent need for a targeted, high-specificity eradication drug. Gastric infection by H. pylori depends on the expression of a nickel-dependent urease in the cytoplasm of the bacteria. Here, we report the 2.0 Å resolution structure of the 1.1 MDa urease in complex with an inhibitor by cryo-electron microscopy and compare it to a β-mercaptoethanol-inhibited structure at 2.5 Å resolution. The structural information is of sufficient detail to aid in the development of inhibitors with high specificity and affinity.
Microbial rhodopsins are light-activated, seven-α-helical, retinylidene transmembrane proteins that have been identified in thousands of organisms across archaea, bacteria, fungi, and algae. Although ...they share a high degree of sequence identity and thus similarity in structure, many unique functions have been discovered and characterized among them. Some function as outward proton pumps, some as inward chloride pumps, whereas others function as light sensors or ion channels. Unique among the microbial rhodopsins characterized thus far, Anabaena sensory rhodopsin (ASR) is a photochromic sensor that interacts with a soluble 14-kDa cytoplasmic transducer that is encoded on the same operon. The sensor itself stably interconverts between all-trans-15-anti and 13-cis-15-syn retinal forms depending on the wavelength of illumination, although only the former participates in a photocycle with a signaling M intermediate. A mutation in the cytoplasmic half-channel of the protein, replacing Asp217 with Glu (D217E), results in the creation of a light-driven, single-photon, inward proton transporter. We present the 2.3 Å structure of dark-adapted D217E ASR, which reveals significant changes in the water network surrounding Glu217, as well as a shift in the carbon backbone near retinal-binding Lys210, illustrating a possible pathway leading to the protonation of Glu217 in the cytoplasmic half-channel, located 15 Å from the Schiff base. Crystallographic evidence for the protonation of nearby Glu36 is also discussed, which was described previously by Fourier transform infrared spectroscopy analysis. Finally, two histidine residues near the extracellular surface and their possible role in proton uptake are discussed.
Light-oxygen-voltage (LOV) domains are blue light-activated signaling modules integral to a wide range of photosensory proteins. Upon illumination, LOV domains form internal protein-flavin adducts ...that generate conformational changes which control effector function. Here we advance our understanding of LOV regulation with structural, biophysical, and biochemical studies of EL222, a light-regulated DNA-binding protein. The dark-state crystal structure reveals interactions between the EL222 LOV and helix-turn-helix domains that we show inhibit DNA binding. Solution biophysical data indicate that illumination breaks these interactions, freeing the LOV and helix-turn-helix domains of each other. This conformational change has a key functional effect, allowing EL222 to bind DNA in a light-dependent manner. Our data reveal a conserved signaling mechanism among diverse LOV-containing proteins, where light-induced conformational changes trigger activation via a conserved interaction surface.
Homologous to bacteriorhodopsin and even more to proteorhodopsin, xanthorhodopsin is a light-driven proton pump that, in addition to retinal, contains a noncovalently bound carotenoid with a function ...of a light-harvesting antenna. We determined the structure of this eubacterial membrane protein-carotenoid complex by X-ray diffraction, to 1.9-Å resolution. Although it contains 7 transmembrane helices like bacteriorhodopsin and archaerhodopsin, the structure of xanthorhodopsin is considerably different from the 2 archaeal proteins. The crystallographic model for this rhodopsin introduces structural motifs for proton transfer during the reaction cycle, particularly for proton release, that are dramatically different from those in other retinal-based transmembrane pumps. Further, it contains a histidine-aspartate complex for regulating the pKa of the primary proton acceptor not present in archaeal pumps but apparently conserved in eubacterial pumps. In addition to aiding elucidation of a more general proton transfer mechanism for light-driven energy transducers, the structure defines also the geometry of the carotenoid and the retinal. The close approach of the 2 polyenes at their ring ends explains why the efficiency of the excited-state energy transfer is as high as almost equal to45%, and the 46° angle between them suggests that the chromophore location is a compromise between optimal capture of light of all polarization angles and excited-state energy transfer.
Half the world's population is chronically infected with Helicobacter pylori, causing gastritis, gastric ulcers and an increased incidence of gastric adenocarcinoma. Its proton-gated inner-membrane ...urea channel, HpUreI, is essential for survival in the acidic environment of the stomach. The channel is closed at neutral pH and opens at acidic pH to allow the rapid access of urea to cytoplasmic urease. Urease produces NH(3) and CO(2), neutralizing entering protons and thus buffering the periplasm to a pH of roughly 6.1 even in gastric juice at a pH below 2.0. Here we report the structure of HpUreI, revealing six protomers assembled in a hexameric ring surrounding a central bilayer plug of ordered lipids. Each protomer encloses a channel formed by a twisted bundle of six transmembrane helices. The bundle defines a previously unobserved fold comprising a two-helix hairpin motif repeated three times around the central axis of the channel, without the inverted repeat of mammalian-type urea transporters. Both the channel and the protomer interface contain residues conserved in the AmiS/UreI superfamily, suggesting the preservation of channel architecture and oligomeric state in this superfamily. Predominantly aromatic or aliphatic side chains line the entire channel and define two consecutive constriction sites in the middle of the channel. Mutation of Trp 153 in the cytoplasmic constriction site to Ala or Phe decreases the selectivity for urea in comparison with thiourea, suggesting that solute interaction with Trp 153 contributes specificity. The previously unobserved hexameric channel structure described here provides a new model for the permeation of urea and other small amide solutes in prokaryotes and archaea.
Although rabbit antibodies are widely used in research, no structures of rabbit antigen-binding fragments (Fab) have been reported. M204 is a rabbit monoclonal antibody that recognizes a generic ...epitope that is common to prefibrillar amyloid oligomers formed from many different amyloidogenic sequences. Amyloid oligomers are widely suspected to be a primary causative agent of pathogenesis in several age-related neurodegenerative diseases, such as Alzheimer's disease. The detailed structure of these amyloid oligomers is not known nor is the mechanism for the recognition of the generic epitope by conformation-dependent monoclonal antibodies.
As a first approach to understanding the mechanism of conformation-dependent antibody recognition, we have crystallized the Fab of M204.
We have determined the structure of the Fab of M204 at 1.54Å resolution. The crystal structure reveals details of the M204 antigen combining site and features unique to rabbit Fabs such as an interdomain disulfide bond on its light chain.
Based on the structural features of the antigen-combining site of the M204, we rule out a “steric zipper” formation, as found in numerous amyloid fibril structures, as a mechanism of antibody-antigen recognition. The details of the first rabbit immunoglobulin Fab structure might also be useful for exploiting the potential of rabbit monoclonal antibodies for the development of humanized rabbit antibodies as therapeutic agents.
► First rabbit Fab crystal structure ► First structure of a conformation-dependent, amyloid oligomer specific antibody ► Structure shows disulfide bonds unique to rabbit IgG. ► The data rule out steric zipper formation as the Fab antigen-binding mechanism.
The atomic structure of the light-driven ion pump bacteriorhodopsin and the surrounding lipid matrix was determined by X-ray diffraction of crystals grown in cubic lipid phase. In the extracellular ...region, an extensive three-dimensional hydrogen-bonded network of protein residues and seven water molecules leads from the buried retinal Schiff base and the proton acceptor Asp85 to the membrane surface. Near Lys216 where the retinal binds, transmembrane helix G contains a π-bulge that causes a non-proline? kink. The bulge is stabilized by hydrogen-bonding of the main-chain carbonyl groups of Ala215 and Lys216 with two buried water molecules located between the Schiff base and the proton donor Asp96 in the cytoplasmic region. The results indicate extensive involvement of bound water molecules in both the structure and the function of this seven-helical membrane protein. A bilayer of 18 tightly bound lipid chains forms an annulus around the protein in the crystal. Contacts between the trimers in the membrane plane are mediated almost exclusively by lipids.
Vitellogenin (Vg) has been implicated as a central protein in the immunity of egg‐laying animals. Studies on a diverse set of species suggest that Vg supports health and longevity through binding to ...pathogens. Specific studies of honey bees (Apis mellifera) further indicate that the vitellogenin (vg) gene undergoes selection driven by local pathogen pressures. Determining the complete 3D structure of full‐length Vg (flVg) protein will provide insights regarding the structure–function relationships underlying allelic variation. Honey bee Vg has been described in terms of function, and two subdomains have been structurally described, while information about the other domains is lacking. Here, we present a structure prediction, restrained by experimental data, of flVg from honey bees. To achieve this, we performed homology modeling and used AlphaFold before using a negative‐stain electron microscopy map to restrict, orient, and validate our 3D model. Our approach identified a highly conserved Ca2+‐ion‐binding site in a von Willebrand factor domain that might be central to Vg function. Thereafter, we used rigid‐body fitting to predict the relative position of high‐resolution domains in a flVg model. This mapping represents the first experimentally validated full‐length protein model of a Vg protein and is thus relevant for understanding Vg in numerous species. Our results are also specifically relevant to honey bee health, which is a topic of global concern due to rapidly declining pollinator numbers.
We describe, for the first time, a full‐length protein structure of honey bee vitellogenin (Vg) using homology modeling, AlphaFold, and a negative stain electron microscopy map. Revealing the oligomeric arrangement and composition of the functional domains around the binding cavities, brings us closer to understanding the molecular mechanisms for Vg's functional role in insect immunity.
In 1993, Huber and co-workers published the structure of an N-terminally truncated version of human annexin A1 lacking the first 32 amino acid residues (PDB code: 1AIN). In 2001, we reported the ...structure of full-length porcine annexin A1 including the N-terminal domain in the absence of calcium ions (PDB code: 1HM6). The latter structure did not reflect a typical annexin core fold, but rather a surprising interaction of the N-terminal domain and the core domain. Comparing these two structures revealed that in the full-length structure the first 12 residues of the N-terminal domain insert into the core of the protein, thereby replacing and unwinding one of the α-helices (helix D in repeat 3) that is involved in calcium binding. We hypothesized that this structure in the absence of calcium ions represents the inactive form of the protein. Furthermore, we proposed that upon calcium binding, the N-terminal domain would be expelled from the core domain and that the core D-helix would reform in the proper conformation for calcium coordination. Herein, we report the X-ray structure of full-length porcine annexin A1 in the presence of calcium. This new structure shows a typical annexin core structure as we hypothesized, with the D-helix back in place for calcium coordination while parts of the now exposed N-terminal domain are disordered. We could locate eight calcium ions in this structure, two of which are octa-coordinated and two of which were not observed in the structure of the N-terminally truncated annexin A1. Possible implications of this calcium-induced conformational switch for the membrane aggregation properties of annexin A1 will be discussed.
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