Crystal Structure of a Yeast Aquaporin at 1.15 Å Reveals a Novel Gating Mechanism

Aquaporins are transmembrane proteins that facilitate the flow of water through cellular membranes. An unusual characteristic of yeast aquaporins is that they frequently contain an extended N terminus of unknown function. Here we present the X-ray structure of the yeast aquaporin Aqy1 from Pichia pastoris at 1.15 Å resolution. Our crystal structure reveals that the water channel is closed by the N terminus, which arranges as a tightly wound helical bundle, with Tyr31 forming H-bond interactions to a water molecule within the pore and thereby occluding the channel entrance. Nevertheless, functional assays show that Aqy1 has appreciable water transport activity that aids survival during rapid freezing of P. pastoris. These findings establish that Aqy1 is a gated water channel. Mutational studies in combination with molecular dynamics simulations imply that gating may be regulated by a combination of phosphorylation and mechanosensitivity.     

The Crystal Structure and RNA-Binding of an Orthomyxovirus Nucleoprotein

Genome packaging for viruses with segmented genomes is often a complex problem. This is particularly true for influenza viruses and other orthomyxoviruses, whose genome consists of multiple negative-sense RNAs encapsidated as ribonucleoprotein (RNP) complexes. To better understand the structural features of orthomyxovirus RNPs that allow them to be packaged, we determined the crystal structure of the nucleoprotein (NP) of a fish orthomyxovirus, the infectious salmon anemia virus (ISAV) (genus Isavirus). As the major protein component of the RNPs, ISAV-NP possesses a bi-lobular structure similar to the influenza virus NP. Because both RNA-free and RNA-bound ISAV NP forms stable dimers in solution, we were able to measure the NP RNA binding affinity as well as the stoichiometry using recombinant proteins and synthetic oligos. Our RNA binding analysis revealed that each ISAV-NP binds ∼12 nts of RNA, shorter than the 24–28 nts originally estimated for the influenza A virus NP based on population average. The 12-nt stoichiometry was further confirmed by results from electron microscopy and dynamic light scattering. Considering that RNPs of ISAV and the influenza viruses have similar morphologies and dimensions, our findings suggest that NP-free RNA may exist on orthomyxovirus RNPs, and selective RNP packaging may be accomplished through direct RNA-RNA interactions.     

植物的水通道蛋白

对近 3年来植物水通道蛋白的特性、特异性表达、水分运输机制、表达调控、在植物水分关系平衡中的作用以及这一领域研究中的有关问题作了介绍与评述     

Aquaporin gating

An acceleration in the rate at which new aquaporin structures are determined means that structural models are now available for mammalian AQP0, AQP1, AQP2 and AQP4, bacterial GlpF, AqpM and AQPZ, and the plant SoPIP2;1. With an apparent consensus emerging concerning the mechanism of selective water transport and proton extrusion, emphasis has shifted towards the issues of substrate selectivity and the mechanisms of aquaporin regulation. In particular, recently determined structures of plant SoPIP2;1, sheep and bovine AQP0, and Escherichia coli AQPZ provide new insights into the underlying structural mechanisms by which water transport rates are regulated in diverse organisms. From these results, two distinct pictures of 'capping' and 'pinching' have emerged to describe aquaporin gating.     

Crystal Structure Determination ofEscherichia coliClpP Starting from an EM-Derived Mask

Large ATP-dependent proteolytic complexes carry out the majority of intracellular proteolysis. To begin to understand the function of these proteases at a structural level, we have combined the information from a number of biophysical techniques such as electron microscopy (EM), small-angle scattering, and x-ray crystallography. In this study, we exploited the inherent symmetry ofEscherichia coliClpP, the proteolytic component of the ClpAP/XP ATP-dependent protease, to determine its x-ray crystal structure to 2.3-Å resolution starting with a phase set derived from a low-resolution mask obtained from EM and small-angle x-ray scattering analysis. Sevenfold and 14-fold noncrystallographic symmetry averaging facilitated phase extension beyond 20 Å and in combination with mask redetermination and matrix refinement was sufficient for completely determining the structure. The structure of ClpP is a homo-tetradecamer composed of two heptameric rings enclosing a cavity of ≈50 Å in diameter that compartmentalizes the 14 serine proteolytic active sites. Comparison of the ClpP structure with those of the 20S proteasome and HslV reveals a striking example of evolutionary convergence, despite them being unrelated in sequence and fold. Moreover, similarity in their overall architecture suggests a common model for their action.     

Crystal Structure of the PIM2 Kinase in Complex with an Organoruthenium Inhibitor

Background

The serine/threonine kinase PIM2 is highly expressed in human leukemia and lymphomas and has been shown to positively regulate survival and proliferation of tumor cells. Its diverse ATP site makes PIM2 a promising target for the development of anticancer agents. To date our knowledge of catalytic domain structures of the PIM kinase family is limited to PIM1 which has been extensively studied and which shares about 50% sequence identity with PIM2.

Principal Findings

Here we determined the crystal structure of PIM2 in complex with an organoruthenium complex (inhibition in sub-nanomolar level). Due to its extraordinary shape complementarity this stable organometallic compound is a highly potent inhibitor of PIM kinases.

Significance

The structure of PIM2 revealed several differences to PIM1 which may be explored further to generate isoform selective inhibitors. It has also demonstrated how an organometallic inhibitor can be adapted to the binding site of protein kinases to generate highly potent inhibitors.

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Aquaporin Water Channels

Thank you very much. I am humbled, I am delighted; I am honored. This is every scientist’s dream: to give the Nobel Lecture in Stockholm. But I would not be honest if I did not tell you that I am having a little anxiety being on this platform. I have lectured a number of times in Sweden, and I thought I would share with you some events preceding a special lecture that I gave here a few years ago. Arriving at Arlanda Airport, I waited in line at the Pass Control behind a group of businessmen in suits with briefcases. I heard the first in line asked by the control officer to state the purpose of his visit to Sweden. When the man replied “business,” the officer approved and stamped his passport. One at a time, each stepped forward and was asked the same thing; each answered “business” and was approved. Eventually it was my turn, and I was dressed in rumpled clothes after spending the night in the Economy Minus section of an SAS jetliner. The officer asked me the purpose of my visit, and I said “I am here to give the von Euler Lecture at Karolinska Institute.” The officer immediately looked up, stared at me, and asked, “Are you nervous?” At that point I became intensely nervous and said “Yes, I am a little nervous.” The officer looked up again and stated “Well, you should be!“ So if the lecturers look a little nervous, the problem is at Arlanda.     

Crystal Structure of Sodium Deoxyinosine Monophosphate

Abstract

The crystal and molecular structure of sodium deoxyinosine monophosphate (5′-dIMP) has been determined by x-ray crystallographic methods. The crystals belong to orthorhombic space group P2₁2₁2₁, with a = 21.079(5) Å, b = 9.206(3) Å and c = 12.770(6) Å. This deoxynucleotide shows common nucleotide features namely anti conformation about the glycosyl bond, C2′ endo pucker for the deoxyribose sugar and gauche-gauche orientation for the phosphate group. The sodium ion is directly coordinated to the O3′ atom, a feature observed in many crystal structures of sodium salts of nucleotides.     

Crystal Structure of the Human Primase

DNA replication in bacteria and eukaryotes requires the activity of DNA primase, a DNA-dependent RNA polymerase that lays short RNA primers for DNA polymerases. Eukaryotic and archaeal primases are heterodimers consisting of small catalytic and large accessory subunits, both of which are necessary for RNA primer synthesis. Understanding of RNA synthesis priming in eukaryotes is currently limited due to the lack of crystal structures of the full-length primase and its complexes with substrates in initiation and elongation states. Here we report the crystal structure of the full-length human primase, revealing the precise overall organization of the enzyme, the relative positions of its functional domains, and the mode of its interaction with modeled DNA and RNA. The structure indicates that the dramatic conformational changes in primase are necessary to accomplish the initiation and then elongation of RNA synthesis. The presence of a long linker between the N- and C-terminal domains of p58 provides the structural basis for the bulk of enzyme''s conformational flexibility. Deletion of most of this linker affected the initiation and elongation steps of the primer synthesis.     

Crystal Structure of a Full-Length Autotransporter

The autotransporter (AT) secretion mechanism is the most common mechanism for the secretion of virulence factors across the outer membrane (OM) from pathogenic Gram-negative bacteria. In addition, ATs have attracted biotechnological and biomedical interest for protein display on bacterial cell surfaces. Despite their importance, the mechanism by which passenger domains of ATs pass the OM is still unclear. The classical view is that the β-barrel domain provides the conduit through which the unfolded passenger moves, with the energy provided by vectorial folding of the β-strand-rich passenger on the extracellular side of the OM. We present here the first structure of a full-length AT, the esterase EstA from Pseudomonas aeruginosa, at a resolution of 2.5 Å. EstA has a relatively narrow, 12-stranded β-barrel that is covalently attached to the passenger domain via a long, curved helix that occupies the lumen of the β-barrel. The passenger has a structure that is dramatically different from that of other known passengers, with a globular fold that is dominated by α-helices and loops. The arrangement of secondary-structure elements suggests that the passenger can fold sequentially, providing the driving force for passenger translocation. The esterase active-site residues are located at the apical surface of the passenger, at the entrance of a large hydrophobic pocket that contains a bound detergent molecule that likely mimics substrate. The EstA structure provides insight into AT mechanism and will facilitate the design of fusion proteins for cell surface display.     

Architecture and Selectivity in Aquaporins: 2.5 Å X-Ray Structure of Aquaporin Z

Aquaporins are a family of water and small molecule channels found in organisms ranging from bacteria to animals. One of these channels, the E. coli protein aquaporin Z (AqpZ), has been shown to selectively conduct only water at high rates. We have expressed, purified, crystallized, and solved the X-ray structure of AqpZ. The 2.5 Å resolution structure of AqpZ suggests aquaporin selectivity results both from a steric mechanism due to pore size and from specific amino acid substitutions that regulate the preference for a hydrophobic or hydrophilic substrate. This structure provides direct evidence on the molecular mechanisms of specificity between water and glycerol in this family of channels from a single species. It is to our knowledge the first atomic resolution structure of a recombinant aquaporin and so provides a platform for combined genetic, mutational, functional, and structural determinations of the mechanisms of aquaporins and, more generally, the assembly of multimeric membrane proteins.     

Crystal Structure of a Mucus-binding Protein Repeat Reveals an Unexpected Functional Immunoglobulin Binding Activity

Lactobacillus reuteri mucus-binding protein (MUB) is a cell-surface protein that is involved in bacterial interaction with mucus and colonization of the digestive tract. The 353-kDa mature protein is representative of a broadly important class of adhesins that have remained relatively poorly characterized due to their large size and highly modular nature. MUB contains two different types of repeats (Mub1 and Mub2) present in six and eight copies, respectively, and shown to be responsible for the adherence to intestinal mucus. Here we report the 1.8-Å resolution crystal structure of a type 2 Mub repeat (184 amino acids) comprising two structurally related domains resembling the functional repeat found in a family of immunoglobulin (Ig)-binding proteins. The N-terminal domain bears striking structural similarity to the repeat unit of Protein L (PpL) from Peptostreptococcus magnus, suggesting binding in a non-immune Fab-dependent manner. A distorted PpL-like fold is also seen in the C-terminal domain. As with PpL, Mub repeats were able to interact in vitro with a large repertoire of mammalian Igs, including secretory IgA. This hitherto undetected activity is consistent with the current model that antibody responses against commensal flora are of broad specificity and low affinity.     

Crystal Structure of an Aquabirnavirus Particle: Insights into Antigenic Diversity and Virulence Determinism

Infectious pancreatic necrosis virus (IPNV), a pathogen of salmon and trout, imposes a severe toll on the aquaculture and sea farming industries. IPNV belongs to the Aquabirnavirus genus in the Birnaviridae family of bisegmented double-stranded RNA viruses. The virions are nonenveloped with a T=13l icosahedral capsid made by the coat protein VP2, the three-dimensional (3D) organization of which is known in detail for the family prototype, the infectious bursal disease virus (IBDV) of poultry. A salient feature of the birnavirus architecture is the presence of 260 trimeric spikes formed by VP2, projecting radially from the capsid. The spikes carry the principal antigenic sites as well as virulence and cell adaptation determinants. We report here the 3.4-Å resolution crystal structure of a subviral particle (SVP) of IPNV, containing 20 VP2 trimers organized with icosahedral symmetry. We show that, as expected, the SVPs have a very similar organization to the IBDV counterparts, with VP2 exhibiting the same overall 3D fold. However, the spikes are significantly different, displaying a more compact organization with tighter packing about the molecular 3-fold axis. Amino acids controlling virulence and cell culture adaptation cluster differently at the top of the spike, i.e., in a central bowl in IBDV and at the periphery in IPNV. In contrast, the spike base features an exposed groove, conserved across birnavirus genera, which contains an integrin-binding motif. Thus, in addition to revealing the viral antigenic determinants, the structure suggests that birnaviruses interact with different receptors for attachment and for cell internalization during entry.Birnaviruses form a distinct family of double-stranded RNA (dsRNA) viruses infecting vertebrates and invertebrates (18). Aquatic birnaviruses are the most abundant and diverse and are grouped in two separate genera: the Aquabirnavirus genus and the Blosnavirus genus, with infectious pancreatic necrosis virus (IPNV) and blotched snakehead virus (BSNV) (16) as respective type species. A third aquatic birnavirus of unassigned genus, Tellina virus 1, was recently described and found to be phylogenetically distant from the two established genera (40). However, the vast majority of aquatic birnaviruses are antigenically related to IPNV (i.e., belong to the Aquabirnavirus genus), regardless of host species or geographical origin (4, 11, 26, 39). They are implicated as etiological agents of disease in a variety of mollusks and fish species important in aquaculture, causing pathologies such as infectious pancreatic necrosis in salmonids, nephroblastoma and branchionephritis in eels, and gill necrosis in clams (21, 34, 50). Viruses in the Aquabirnavirus genus display considerable antigenic diversity and have substantial differences in biological properties such as host range and optimal replication temperature. These features contrast with the properties of other birnaviruses, in particular those infecting terrestrial species (avibirnaviruses and entomobirnaviruses). Based on reciprocal neutralization tests with polyclonal and monoclonal antibodies, nine cross-reacting serotypes (A1 to A9) have been defined for IPNV and related aquabirnaviruses (26). Serotype A2 (also known as Sp) is the most common serotype found in Europe.Birnavirus particles are nonenveloped, displaying a single-shelled T=13l icosahedral capsid of about 70 nm in diameter, composed of 260 trimers of viral protein 2 (VP2) (5, 15, 42). Internal to the virion are VP3, which forms a ribonucleoprotein complex with the genomic RNA (9, 27, 36), and VP1, the viral RNA-dependent RNA polymerase, which is found both free and covalently attached to the genomic RNA (19). The birnavirus genome consists of two segments of dsRNA. While the smaller segment B has a single open reading frame (ORF) coding for VP1, segment A has two, a large and a small ORF, encoding the polyprotein precursor pVP2-VP4-VP3 and the nonstructural VP5, respectively. VP4 is a protease that cleaves its own N and C termini off the polyprotein, thus also releasing pVP2 (the VP2 precursor) and VP3 within the infected cell (3, 20). Subsequent serial cleavages at the C terminus of pVP2 upon particle assembly yield mature VP2 (amino acids 1 to 442 of the IPNV polyprotein) and three other peptides that remain within the virion (22). The longest of these peptides was shown to destabilize cell membranes, suggesting a role during entry (40). Maturation of the pVP2 precursor during assembly and the presence of VP3 are important for correct morphogenesis of icosahedral, T=13l virus particles.Recombinant expression of mature VP2 alone leads to assembly of small, dodecahedral T=1 subviral particles (SVPs) containing 20 VP2 trimers (10). The crystal structures of the SVPs of infectious bursal disease virus of poultry (IBDV) were reported by several groups (15, 23, 31) as well as the three-dimensional (3D) structure of an intact T=13l IBDV virion (15).Exposed at the virion surface, VP2 displays the humoral antigenic determinants of the virus and is the only viral protein shown to induce protective immunity. Furthermore, VP2 plays a key role during virus entry, being responsible for receptor recognition (8). Key residues controlling virulence and cell culture adaptation of IPNV were thus found to map to VP2 (46).We report here the crystal structure of the IPNV SVP and show that, like its IBDV counterpart, it is composed of 20 VP2 trimers organized with T=1 icosahedral symmetry. IPNV VP2 displays the same fold, and the SVP is organized in the same way, as anticipated from the 43% overall amino acid sequence identity between the VP2s of the two viruses. The molecules differ significantly, however, in the loops of domain P, which forms the projections or spikes. In particular, the 3D structure shows a clustering of variable residues in the outermost loops at the top of domain P. Residues associated with virulence and cell culture adaptation map to the most peripheral of these loops—away from the 3-fold axis at the convex top of VP2. This is in contrast to IBDV, in which virulence determinants map to a central bowl at the top of the VP2 spike. These results provide new insights for understanding the determinism of antigenicity and virulence of Aquabirnavirus strains.     

Crystal Structure of an Unusual Single-Stranded DNA-Binding Protein Encoded by Staphylococcal Cassette Chromosome Elements

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Crystal Structure and Functional Analysis of JMJD5 Indicate an Alternate Specificity and Function

JMJD5 is a Jumonji C (JmjC) protein that has been implicated in breast cancer tumorigenesis, circadian rhythm regulation, embryological development, and osteoclastogenesis. Recently, JMJD5 (also called KDM8) has been reported to demethylate dimethylated Lys-36 in histone H3 (H3K36me2), regulating genes that control cell cycle progression. Here, we report high-resolution crystal structures of the human JMJD5 catalytic domain in complex with the substrate 2-oxoglutarate (2-OG) and the inhibitor N-oxalylglycine (NOG). The structures reveal a β-barrel fold that is conserved in the JmjC family and a long shallow cleft that opens into the enzyme's active site. A comparison with other JmjC enzymes illustrates that JMJD5 shares sequence and structural homology with the asparaginyl and histidinyl hydroxylase FIH-1 (factor inhibiting hypoxia-inducible factor 1 [HIF-1]), the lysyl hydroxylase JMJD6, and the RNA hydroxylase TYW5 but displays limited homology to JmjC lysine demethylases (KDMs). Contrary to previous findings, biochemical assays indicate that JMJD5 does not display demethylase activity toward methylated H3K36 nor toward the other methyllysines in the N-terminal tails of histones H3 and H4. Together, these results imply that JMJD5 participates in roles independent of histone demethylation and may function as a protein hydroxylase given its structural homology with FIH-1 and JMJD6.