L1 Loop Research Articles

Crystal structure of the Eg5 - K858 complex and implications for structure-based design of thiadiazole-containing inhibitors

The thiadiazole scaffold is an important core moiety in a variety of clinical drug candidates targeting a range of diseases. For example, the 2,4,5-substituted 1,3,4-thiadiazole scaffold is present in a lead compound and at least two clinical candidates targeting the human motor protein Eg5, against neoplastic diseases. An inhibitor named K858 has in vivo activity in various mouse xenografts whereas the clinical candidates (S)-ARRY-520 and (R)-Litronesib have entered clinical trials with the former one in phase III clinical trials either alone or in combination with a proteasome inhibitor against relapsed/refractory multiple myeloma. Astonishingly, structural data are lacking for all thiadiazole-containing Eg5 inhibitors. Here we report the structure determination of two crystal forms of the ternary Eg5-ADP-K858 complex, locking the motor in the so-called final inhibitor bound state, thus blocking ADP release, a crucial stage for Eg5 activity. K858 acts at the established allosteric inhibitor-binding pocket formed of helix α2, loop L5 and helix α3. The structure of the complex has far reaching consequences for thiadiazole containing Eg5 inhibitors. For example, we could rationalise the structure-activity relationship in the crucial 5-position of the thiadiazole scaffold and the complex will serve in the future as a basis for strucutre-based drug design.

Histone H2A variants confer specific properties to nucleosomes and impact on chromatin accessibility.

In eukaryotes, variants of core histone H2A are selectively incorporated in distinct functional domains of chromatin and are distinguished by conserved sequences of their C-terminal tail, the L1 loop and the docking domain, suggesting that each variant confers specific properties to the nucleosome. Chromatin of flowering plants contains four types of H2A variants, which biochemical properties have not been characterized. We report that in contrast with animals, in Arabidopsis thaliana H2A variants define only four major types of homotypic nucleosomes containing exclusively H2A, H2A.Z, H2A.X or H2A.W. In vitro assays show that the L1 loop and the docking domain confer distinct stability of the nucleosome. In vivo and in vitro assays suggest that the L1 loop and the docking domain cooperate with the C-terminal tail to regulate chromatin accessibility. Based on these findings we conclude that the type of H2A variant in the nucleosome impacts on its interaction with DNA and propose that H2A variants regulate the dynamics of chromatin accessibility. In plants, the predominance of homotypic nucleosomes with specific physical properties and their specific localization to distinct domains suggest that H2A variants play a dominant role in chromatin dynamics and function.

Conformational Dynamics of thiM Riboswitch To Understand the Gene Regulation Mechanism Using Markov State Modeling and the Residual Fluctuation Network Approach.

Thiamine pyrophosphate (TPP) riboswitch is a cis-regulatory element in the noncoding region of mRNA. The aptamer domain of TPP riboswitch detects the high abundance of coenzyme thiamine pyrophosphate (TPP) and modulates the gene expression for thiamine synthetic gene. The mechanistic understanding in recognition of TPP in aptamer domain and ligand-induced compactness for folding of expression platform are most important to designing novel modulators. To understand the dynamic behavior of TPP riboswitch upon TPP binding, molecular dynamics simulations were performed for 400 ns in both apo and TPP bound forms of thiM riboswitch from E. coli and analyzed in terms of eRMSD-based Markov state modeling and residual fluctuation network. Markov state models show good correlations in transition probability among metastable states from simulated trajectory and generated models. Structural compactness in TPP bound form is observed which is correlated with SAXS experiment. The importance of junction of P4 and P5 is evident during dynamics, which correlates with FRET analysis. The dynamic nature of two sensor forearms is due to the flexible P1 helix, which is its intrinsic property. The transient state in TPP-bound form was observed in the Markov state model, along with stable states. We believe that this transient state is responsible to assist the influx and outflux of ligand molecule by creating a solvent channel around the junction region of P4 and P5 and such a structure was anticipated in FRET analysis. The dynamic nature of riboswitch is dependent on the interaction between residues on distal loops L3 and L5/P3 and junction P4 and P5, J3/2 which stabilize the J2/4. It helps in the transfer of allosteric information between J2/4 and P3/L5 tertiary docking region through the active site residues. Understanding such information flow will benefit in highlighting crucial residues in highly dynamic and kinetic systems. Here, we report the residues and segments in riboswitch that play vital roles in providing stability and this can be exploited in designing inhibitors to regulate the functioning of riboswitches.

A consensus-guided approach yields a heat-stable alkane-producing enzyme and identifies residues promoting thermostability

Aldehyde-deformylating oxygenase (ADO) is an essential enzyme for production of long-chain alkanes as drop-in biofuels, which are compatible with existing fuel systems. The most active ADOs are present in mesophilic cyanobacteria, especially Nostoc punctiforme Given the potential applications of thermostable enzymes in biorefineries, here we generated a thermostable (Cts)-ADO based on a consensus of ADO sequences from several thermophilic cyanobacterial strains. Using an in silico design pipeline and a metagenome library containing 41 hot-spring microbial communities, we created Cts-ADO. Cts-ADO displayed a 3.8-fold increase in pentadecane production on raising the temperature from 30 to 42 °C, whereas ADO from N. punctiforme (Np-ADO) exhibited a 1.7-fold decline. 3D structure modeling and molecular dynamics simulations of Cts- and Np-ADO at different temperatures revealed differences between the two enzymes in residues clustered on exposed loops of these variants, which affected the conformation of helices involved in forming the ADO catalytic core. In Cts-ADO, this conformational change promoted ligand binding to its preferred iron, Fe2, in the di-iron cluster at higher temperature, but the reverse was observed in Np-ADO. Detailed mapping of residues conferring Cts-ADO thermostability identified four amino acids, which we substituted individually and together in Np-ADO. Among these substitution variants, A161E was remarkably similar to Cts-ADO in terms of activity optima, kinetic parameters, and structure at higher temperature. A161E was located in loop L6, which connects helices H5 and H6, and supported ligand binding to Fe2 at higher temperatures, thereby promoting optimal activity at these temperatures and explaining the increased thermostability of Cts-ADO.

An auto-inhibitory helix in CTP:phosphocholine cytidylyltransferase hijacks the catalytic residue and constrains a pliable, domain-bridging helix pair

The activity of CTP:phosphocholine cytidylyltransferase (CCT), a key enzyme in phosphatidylcholine synthesis, is regulated by reversible interactions of a lipid-inducible amphipathic helix (domain M) with membrane phospholipids. When dissociated from membranes, a portion of the M domain functions as an auto-inhibitory (AI) element to suppress catalysis. The AI helix from each subunit binds to a pair of α helices (αE) that extend from the base of the catalytic dimer to create a four-helix bundle. The bound AI helices make intimate contact with loop L2, housing a key catalytic residue, Lys122 The impacts of the AI helix on active-site dynamics and positioning of Lys122 are unknown. Extensive MD simulations with and without the AI helix revealed that backbone carbonyl oxygens at the point of contact between the AI helix and loop L2 can entrap the Lys122 side chain, effectively competing with the substrate, CTP. In silico, removal of the AI helices dramatically increased αE dynamics at a predicted break in the middle of these helices, enabling them to splay apart and forge new contacts with loop L2. In vitro cross-linking confirmed the reorganization of the αE element upon membrane binding of the AI helix. Moreover, when αE bending was prevented by disulfide engineering, CCT activation by membrane binding was thwarted. These findings suggest a novel two-part auto-inhibitory mechanism for CCT involving capture of Lys122 and restraint of the pliable αE helices. We propose that membrane binding enables bending of the αE helices, bringing the active site closer to the membrane surface.

The identification of discontinuous epitope in the human cystatin C – Monoclonal antibody HCC3 complex

Human cystatin C (hCC) is a cysteine proteinase inhibitor involved in pathophysiological processes of dimerization and amyloid formation. These processes are directly associated with a number of neurodegenerative disorders such as Alzheimer disease or hereditary cystatin C amyloid angiopathy (HCCAA). One of the ideas on how to prevent amyloid formation is to use immunotherapy. HCC3 is one of a group of antibodies binding to hCC and reducing the in vitro formation of cystatin C dimers. Therefore, identification of the binding sites in the hCC-HCC3 complex may facilitate a search of effective drugs against HCCAA as well as understanding the mechanisms of neurodegenerative disorders.In this work we present epitope identification of the hCC-HCC3 complex using methods such as affinity chromatography, epitope excision and extraction MS approach, enzyme-linked immunosorbent assay and hydrogen-deuterium exchange mass spectrometry (HDX MS). Comprehensive analysis of the obtained results allowed us to identify the epitope sequence with the key fragment covering hCC L1 loop and two potential epitopic fragments – α-helical part, hCC (17–28) and β4 strand in C-terminal part of hCC. The presence of the L1 loop in the epitope sequence accounts for the significant reduction of hCC dimer formation in the presence of HCC3 antibody. Significance of the studyDeciphering the mechanism of the cystatin C aggregation process and detailed analysis of the interactions between hCC, or its pathogenic variant, and monoclonal antibodies, potentially constituting aggregation inhibitors, might be of great value as there still is a complete lack of any kind of efficient therapy for young people with the pathogenic mutation of hCC.

Native State Organization of Outer Membrane Porins Unraveled by HDx-MS.

Hydrogen-deuterium exchange (HDx) associated with mass spectrometry (MS) is emerging as a powerful tool to provide conformational information about membrane proteins. Unfortunately, as for X-ray diffraction and NMR, HDx performed on reconstituted in vitro systems might not always reflect the in vivo environment. Outer-membrane vesicles naturally released by Escherichia coli were used to carry out analysis of native OmpF through HDx-MS. A new protocol compatible with HDx analysis that avoids hindrance from the lipid contents was setup. The extent of deuterium incorporation was in good agreement with the X-ray diffraction data of OmpF as the buried β-barrels incorporated a low amount of deuterium, whereas the internal loop L3 and the external loops incorporated a higher amount of deuterium. Moreover, the kinetics of incorporation clearly highlights that peptides segregate well in two distinct groups based exclusively on a trimeric organization of OmpF in the membrane: peptides presenting fast kinetics of labeling are facing the complex surrounding environment, whereas those presenting slow kinetics are located in the buried core of the trimer. The data show that HDx-MS applied to a complex biological system is able to reveal solvent accessibility and spatial arrangement of an integral outer-membrane protein complex.

Mechanism of An Anticancer Peptide Rescuing p53 from Degradation by COP1

A 28 amino‐acid peptide derived from azurin (amino acids 50–77, p28) preferentially enters human cancer cells and induces apoptosis or growth inhibition. Previous studies have revealed that p28 exerts its anticancer activity mainly by interacting with the p53 DNA binding domain (DBD) and interfering in the p53 degradation signaling pathway. However, the interacting mechanisms between p28 and p53‐DBD are missing and how p28 inhibits p53‐DBD ubiquitination remains largely unknown. In this work, we characterized the interacting mechanisms between p28 and p53‐DBD by a combination of NMR, X‐ray crystallography, ITC and computer simulations. Interactions between p28 and p53‐DBD were mainly mediated by hydrophobic contacts. Structural models of the p28/p53‐DBD complex obtained with NMR datadriven docking indicated that p28 adopted an α‐helix upon binding to p53‐DBD. Val59, Val60, Thr61, Tyr72 and Leu73 of p28 made extensive interactions with the L1 loop and S7–S8 loop of p53‐DBD. Site‐directed mutagenesis and ITC characterization showed that single alanine substitution of the above mentioned hydrophobic residues reduced the binding affinity by more than one order of magnitude. GST pull‐down assays showed that the interactions between p53‐ DBD and the E3 ligase COP1 were blocked by p28. Since alanine substitution of key hydrophobic residues reduced the binding affinity of p28 with p53‐DBD, alanine mutated p28 were less efficient in blocking the p53‐DBD/COP1 interaction. Determination of the p28/p53‐ DBD complex structure is in progress, which will allow us to rationally design p28 derived peptides exhibiting higher affinity with p53‐DBD and stronger anticancer activity in the future.Support or Funding InformationThis work was supported by National Natural Science Foundation of China (Grant number: 21603121, Y.H.) and Hubei University of Technology (M. G. and Y.H.).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Caspase‐6 Self‐Activation Enables Distinct Helix‐Strand Interconversion Upon Substrate Binding

Caspases are cysteine proteases that are major players in key cellular processes, including apoptosis and inflammation. Amongst the human caspases, caspase‐6 has been implicated in playing a unique and critical role in the neurodegenerative pathways of Alzheimer's, Huntington's and Parkinson's disease. Unfortunately, structural similarities between caspase‐6 and other caspases have hampered precise targeting of caspase‐6 uniquely. All caspases can exist in a canonical conformation, in which the substrate binds atop a beta‐strand platform in the 130's region. This caspase‐6 region can also adopt a helical conformation that has not been seen in any other caspases. We have shown that caspase‐6 is inherently and dramatically more conformationally dynamic than closely related caspase‐7. In contrast to caspase‐7, which rests constitutively in the strand conformation before and after substrate binding, hydrogen/deuterium exchange data for the L2’ and 130's regions suggested that prior to substrate binding, caspase‐6 exists in a dynamic equilibrium between the helix and strand conformations. Caspase‐6 transitions exclusively to the canonical strand conformation only upon substrate binding. Glu‐135, which showed a noticeably different calculated pKas in the helix and strand conformations, appears to play a key role in the interconversion between the helix and strand conformations. We have also mapped the local changes in the conformational flexibility of procaspase‐6 at the discrete states that reflect series of cleavage events that ultimately lead to the fully active, substrate‐bound state. Intramolecular self‐cleavage at Asp‐193 evoked higher solvent exposure in the regions of the substrate binding loops L1, L3, L4 and in the 130's, the intersubunit linker region, and the 26–32 region, as well as stabilized loop 2. Further removal of the linker allowed caspase‐6 to gain more flexibility in the 130's region and in the L2 region converting caspase‐6 to a competent substrate‐binding state. The prodomain region was found to be intrinsically disordered independent of the activation step of caspase‐6; however, its complete removal resulted in the protection of the adjacent 26–32 region, suggesting that this region may play a regulatory role, likely as an exosite for substrate recognition. The molecular details of caspase‐6 dynamics in solution provide a comprehensive scaffold for strategic design of therapeutic approaches for neurodegenerative disorders.Support or Funding InformationThis work was supported by the National Institutes of Health (NIH) GM 080532.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Three Hcp homologs with divergent extended loop regions exhibit different functions in avian pathogenic Escherichia coli

Type VI secretion systems (T6SSs) contribute to the pathogenicity of avian pathogenic Escherichia coli (APEC), one of the leading causative agents of sepsis and meningitis in poultry. The Hcp protein is a core component of the T6SS tail tube and acts as an exported receptor and a chaperone of effectors. In this study, four distinct Hcp types (Ia, Ib, IIa, and IIb) were designated in Gram-negative bacteria, three of which were widely distributed in APEC. We detected divergence in transcription levels among three hcp clusters in 50% duck serum and demonstrated that hcp1 was upregulated by relieving Fur repression. Further analyses revealed that the host serum could activate the hcp2B operon by H-NS derepression to transcribe the downstream xmtU/xmtV pair for inter-bacterial antagonism. Notably, in a structural analysis based on the genetic classification, Hcp proteins exhibited significant differences in the extended loop regions, suggesting that these regions were related to their functional properties. Indeed, the variant region Vs2 (Loop L2, 3) in Hcp1 and Hcp2B was essential for the delivery of antibacterial effectors and the inhibition of macrophage phagocytosis. Further analyses using a duck model indicated that these Hcps play different roles in the pathogenic processes of APEC and immunoprotection. These results indicated that the functional differentiation of Hcp homologs was driven by differences in transcriptional regulation, extended loop regions, and effector delivery.

Synthesis and physicochemical studies of amyloidogenic hexapeptides derived from human cystatin C.

Human cystatin C (hCC) is a low molecular mass protein that belongs to the cystatin superfamily. It is an inhibitor of extracellular cysteine proteinases, present in all human body fluids. At physiological conditions, hCC is a monomer, but it has a tendency to dimerization. Naturally occurring hCC mutant, with leucine in position 68 substituted by glutamine (L68Q), is directly involved in the formation of amyloid deposits, independently of other proteins. This process is the primary cause of hereditary cerebral amyloid angiopathy, observed mainly in the Icelandic population. Oligomerization and fibrillization processes of hCC are not explained equally well, but it is proposed that domain swapping is involved in both of them. Research carried out on the fibrillization process led to new hypothesis about the existence of a steric zipper motif in amyloidogenic proteins. In the hCC sequence, there are 2 fragments which may play the role of a steric zipper: the loop L1 region and the C-terminal fragment. In this work, we focused on the first of these. Nine hexapeptides covering studied hCC fragment were synthesized, and their fibrillogenic potential was assessed using an array of biophysical methods. The obtained results showed that the studied hCC fragment has strong profibrillogenic propensities because it contains 2 fragments fulfilling the requirements for an effective steric zipper located next to each other, forming 1 super-steric zipper motif. This hCC fragment might therefore be responsible for the enhanced amyloidogenic properties of dimeric or partially unfolded hCC.

Molecular Determinants for 23S rRNA Recognition and Modification by the E. coli Pseudouridine Synthase RluE

The isomerization of uridine to pseudouridine is the most common type of RNA modification found in RNAs across all domains of life and is performed by RNA-dependent and RNA-independent enzymes. The Escherichia coli pseudouridine synthase RluE acts as a stand-alone, highly specific enzyme forming the universally conserved pseudouridine at position 2457, located in helix 89 (H89) of the 23S rRNA in the peptidyltransferase center. Here, we conduct a detailed structure–function analysis to determine the structural elements both in RluE and in 23S rRNA required for RNA–protein interaction and pseudouridine formation. We determined that RluE recognizes a large part of 23S rRNA comprising both H89 and the single-stranded flanking regions which explains the high substrate specificity of RluE. Within RluE, the target RNA is recognized through sequence-specific contacts with loop L7–8 as well as interactions with loop L1–2 and the flexible N-terminal region. We demonstrate that RluE is a faster pseudouridine synthase than other enzymes which likely enables it to act in the early stages of ribosome formation. In summary, our biochemical characterization of RluE provides detailed insight into the molecular mechanism of RluE forming a highly conserved pseudouridine during ribosome biogenesis.

Identification of inhibitor against H. pylori HtrA protease using structure-based virtual screening and molecular dynamics simulations approaches

The HtrA protease of Helicobacter pylori, which efficiently colonizes at the gastric epithelial of host cells, disrupts the mucosal integrity of E-cadherin and spreads inflammatory diseases including gastric cancer by cleaving the cell-cell adhesion of the host. The lack of knowledge on the molecular diversity, structural and functional behavior of HpHtrA necessitated the present study to explore its inhibition mechanism. At first, the similarity of HpHtrA with other gastro-intestinal pathogenic HtrA bacteria and its remote relationship with the Human HtrA homologs were ensured by the phylogenetic analysis and hence was identified as a novel therapeutic target for further design of inhibitors. The three dimensional structure of HpHtrA was modeled and simulated to achieve its stable conformation and was used as a receptor to screen for the possible lead compound through virtual screening (using ∼ 1.3 million compounds). Molecular dynamics simulations followed by the binding energy analysis revealed the affinity of the compound 300040 in forming a stable complex with HpHtrA and thereby revealed its potent role in inhibiting HpHtrA. It is also worthy to mention that, structurally, the ligand binding at the catalytic site of HpHtrA is mainly facilitated by the significant dynamics of L2 loop. Based on the present study, the hydroxyl-piperidine with 4-aminopiperidine scaffold is proposed to be one of the best possible lead compounds for the inhibition of H. pylori.

Molecular epidemiology and phylogenetic analysis of fowl adenoviruses caused hydropericardium outbreak in China during 2015

An outbreak of hydropericardium syndrome has been ongoing in East China since 2015. To decipher the disease etiology, 26 full-length sequences of the adenovirus hexon gene were cloned and sequenced. Phylogenetic trees and sequence alignments revealed that all 26 strains belonged to subgroup C serotype 4 and shared some consistent molecular characteristics that showed only 9 mutations without amino acid (AA) deletions. Eight sites were located in L1 and one in L2. To determine whether the complete hexon gene was under positive selection pressure, site-by-site analysis was performed to identify specific codons that underwent evolutionary selection. Analysis identified 7 positively selected codons in L1 (AA 164, 168, 195, and 243) and L2 (AA 379, 402, and 408). The other 2 sites (AA 140 and 680) were located in P1 and P2, respectively. The results reveal that hexon loops L1 and L2 are hypervariable regions. Moreover, elucidation of viral distribution indicated that the liver is a major target organ, and FAdV-4 is a pantropic virus. In summary, FAdV-4 has been identified as an epidemic strain, and viral evolution is ongoing; thus, continuous surveillance of this virus will contribute to the control of future epidemics.

The structurally similar TRFH domain of TRF1 and TRF2 dimers shows distinct behaviour towards TIN2

The telomere repeat binding-factor 1 and 2 (TRF1 and TRF2) proteins of the shelterin complex bind to duplex telomeric DNA as homodimers, and the homodimerization is mediated by their TRFH (TRF-homology) domains. We performed molecular dynamic (MD) simulations of the dimer forms of TRF1TRFH and TRF2TRFH in the presence/absence of the TIN2TBM (TIN2, TRF-interacting nuclear protein 2, TBM, TRF-binding motif) peptide. The MD results suggest that TIN2TBM is necessary to ensure the stability of TRF1TRFH homodimer but not the TRF2TRFH homodimer. In TRF1-TIN2-TRF2 complex, the peptide enhances the protein-protein interactions to yield a stable heterodimer. Both monomers in TRF1TRFH homodimer interact almost equally with the peptide, whereas in TRF2TRFH homodimer, monomer TRF2TRFH(M1) exhibits more dominant interactions than the TRF2TRFH(M2). The common residues of TRF1/2TRFH(M1) that form interactions with TIN2TBM in all peptide-bound systems originate from the H3 (helix) and L3 (loop) regions. Additionally, in the homodimer systems, residues of TRF1/2TRFH(M2) also interact with the peptide. The residue pair E71-K213 is responsible for different conformations of TRF1TRFH homodimers; specifically, this residue pair enhances the protein-peptide/protein interactions in peptide-bound/unbound systems, respectively. TRF1TRFH and TRF2TRFH proteins have a conserved but different interface responsible for the protein-protein/peptide interactions that exist in the corresponding dimers.

Effects of Hydroxylation at Proline 567 in HIF-1α on the Binding to pVHL

Hypoxia is a physiological condition commonly present in tissue tumor growth and hypoxia training. Hypoxia inducible factor-1α (HIF-1α) plays an essential role in the regulation of hypoxia in animal cells. This mechanism is mediated by the von Hippel–Lindau tumor suppressor protein (pVHL) through the hydroxylation of HIF-1α proline 564. However, previous study has shown proline 567 was hydroxylated by prolyl hydroxylase-3 (PHD-3) in vitro. In addition, recent computational work has illustrated that a new hydrogen bond network localized around HYP567 was rearranged. In this work, we performed all-atom molecular dynamics (MD) simulations to investigate the structural effect and the interaction between pVHL and HIF-1α in status of non-hydroxylation proline (noHYP), hydroxylation proline 564 (HYP564), hydroxylation proline 567 (HYP567), and hydroxylation prolines 564 and 567(2HYP). Our cumulative 5 μs MD simulations reveal that HYP564 shows a lower RMSD value than the rest of variations, while 2HYP increases the structural flexibility of loop L7 (V137-P146) and α-domain (V155-P192) in pVHL comparing with HYP564. Hydrogen bond profile analysis illustrates that both HYP564 and 2HYP variations have two more hydrogen bonds (HYP564-HIS115 and SER111-HYP564) presented in an extremely steady condition, while 2HYP slightly reduces the intensity of H-bonds than HYP564. After the binding free energy calculation, HIF-1α shows a strong binding affinity to pVHL in HYP564 system, while the 2HYP obviously decreases the binding free energy between HIF-1α peptide and pVHL. In addition, our study highlights the conformational dynamics process between pVHL and variations of HIF-1α, suggesting that the hydroxylation of proline 567 may disturb the capture of HIF-1α by pVHL. Considering other evidences, we suggest that proline 567 in HIF-1α may not be hydroxylated in humans, at least in normal condition.

Local and Global Dynamics in Klebsiella pneumoniae Outer Membrane Protein a in Lipid Bilayers Probed at Atomic Resolution

The role of membrane proteins in cellular mechanisms strongly depends on their dynamics; herein we make use of advances in 1H-detected MAS NMR to describe the dynamics of the membrane domain of the Outer membrane protein A of Klebsiella pneumoniae (KpOmpA). By measuring 1H-15N dipolar-coupling as well as 15N R1 and R1ρ relaxation rates at fast (60 kHz) MAS and high magnetic field (1 GHz), we were able to describe the motion of the β-barrel as a collective rocking of low amplitude and of hundreds of nano-seconds time scale. Residual local motions at the edges of the strands, underscored by enhanced 15N R1ρ relaxation rates, report on the mobility of the connected loops. In agreement with MAS NMR data, proteolysis experiments performed on the full length KpOmpA as well as on its membrane domain, reconstituted in liposomes or in detergent micelles, revealed in all cases the existence of a unique Trypsin cleavage site within the membrane domain (out of 16 potential Lys and Arg sites). This site is located in the extracellular loop L3, indicating that this loop may be highly accessible to protein-protein interactions. KpOmpA is involved in cell-cell recognition, adhesion and immune response mechanisms. The L3 region may therefore play a key role in pathogenicity.

Crystal structure of the outer membrane protein OmpU from Vibrio cholerae at 2.2 Å resolution.

Vibrio cholerae causes a severe disease that kills thousands of people annually. The outer membrane protein OmpU is the most abundant outer membrane protein in V. cholerae, and has been identified as an important virulence factor that is involved in host-cell interaction and recognition, as well as being critical for the survival of the pathogenic V. cholerae in the host body and in harsh environments. The mechanism of these processes is not well understood owing to a lack of the structure of V. cholerae OmpU. Here, the crystal structure of the V. cholerae OmpU trimer is reported to a resolution of 2.2 Å. The protomer forms a 16-β-stranded barrel with a noncanonical N-terminal coil located in the lumen of the barrel that consists of residues Gly32-Ser42 and is observed to participate in forming the second gate in the pore. By mapping the published functional data onto the OmpU structure, the OmpU structure reinforces the notion that the long extracellular loop L4 with a β-hairpin-like motif may be critical for host-cell binding and invasion, while L3, L4 and L8 are crucially implicated in phage recognition by V. cholerae.

Hexon and fiber gene changes in an attenuated fowl adenovirus isolate from Malaysia in embryonated chicken eggs and its infectivity in chickens.

Fowl adenovirus (FAdV) is distributed worldwide and causes economic losses in the poultry industry. The objectives of this study were to determine the hexon and fiber gene changes in an attenuated FAdV isolate from Malaysia in specific pathogen-free chicken embryonated eggs (SPF CEE) and its infectivity in commercial broiler chickens. SPF CEE were inoculated with 0.1 mL FAdV inoculum via the chorioallantoic membrane (CAM) for 20 consecutive passages. The isolate at passage 20 (E20), with a virus titer of 108.7TCID50/mL (TCID50, 50% tissue culture infective dose), was inoculated (0.5 mL) into one-day-old commercial broiler chicks either via oral or intraperitoneal routes. The study demonstrated that 100% embryonic mortality was recorded from E2 to E20 with a delayed pattern at E17 onwards. The lesions were confined to the liver and CAM. Substitutions of amino acids in the L1 loop of hexon at positions 49 and 66, and in the knob of fiber at positions 318 and 322 were recorded in the E20 isolate. The isolate belongs to serotype 8b and is non-pathogenic to broiler chickens, but it is able to induce a FAdV antibody titer. It appears that molecular changes in the L1 loop of hexon and the knob of fiber are markers for FAdV infectivity.

Decoding the centromeric nucleosome through CENP-N.

Centromere protein (CENP) A, a histone H3 variant, is a key epigenetic determinant of chromosome domains known as centromeres. Centromeres nucleate kinetochores, multi-subunit complexes that capture spindle microtubules to promote chromosome segregation during mitosis. Two kinetochore proteins, CENP-C and CENP-N, recognize CENP-A in the context of a rare CENP-A nucleosome. Here, we reveal the structural basis for the exquisite selectivity of CENP-N for centromeres. CENP-N uses charge and space complementarity to decode the L1 loop that is unique to CENP-A. It also engages in extensive interactions with a 15-base pair segment of the distorted nucleosomal DNA double helix, in a position predicted to exclude chromatin remodelling enzymes. Besides CENP-A, stable centromere recruitment of CENP-N requires a coincident interaction with a newly identified binding motif on nucleosome-bound CENP-C. Collectively, our studies clarify how CENP-N and CENP-C decode and stabilize the non-canonical CENP-A nucleosome to enforce epigenetic centromere specification and kinetochore assembly.