L1 Loop Research Articles

ID: 208: Comparison of the antiviral activity of Mus spretus Mx1 and Mus musculus A2G Mx1 against influenza A viruses

The interferon-induced Mx1 gene is an important part of the mammalian defense against influenza viruses. Mouse Mx1 inhibits influenza virus replication and transcription by suppressing the polymerase activity of viral ribonucleoproteins (vRNPs). Here, we compared the anti-influenza activity of Mx1 from Mus musculus A2G with its ortholog from Mus spretus . We found that the antiviral activity of M. spretus Mx1 was less potent than that of M. musculus Mx1. Sequence comparison of M. musculus with M. spretus Mx1 revealed 25 amino acid differences, half of which are present in the GTPase domain. However, no differences in GTPase activity could be detected. In addition, 2 differences are present in loop L4. Replacing one of these residues in M. spretus Mx1 by the corresponding residue of A2G Mx1 increased its antiviral activity. We also show that deletion of loop L4 prevented NP binding and hence abolished antiviral activity of mouse Mx1. Taken together, these results indicate that loop L4 of mouse Mx1 is an important determinant of antiviral activity, similar to its human homolog MxA. Our findings suggest that Mx proteins from different mammals use a common mechanism to inhibit influenza A viruses.

Clinical presentation and mutation analysis of VHL disease in a large Chinese family.

Von Hippel-Lindau (VHL) disease is an autosomal dominantly inherited neoplastic disorder characterized by marked phenotypic variability and age-dependent penetrance. This disease is caused by germline mutations in the VHL tumor suppressor gene. Systematic physical examinations, imaging assessments and molecular genetic tests for the VHL gene were performed in a large Chinese VHL family. The examined Chinese VHL family, which has 25 members from four generations, including 7 diagnosed VHL patients and 2 asymptomatic mutation carriers. The average ages of first onset for generations I, II, and III were 37, 30 and 16, respectively. The male:female ratio among VHL patients was 6:1. Molecular genetic investigations detected the c.433C>T [p.Q145X] nonsense mutation in the VHL gene. Molecular modeling of the VHL-ElonginC- ElonginB-HIF-1α complex predicted that the p.Q145X mutation markedly alters the L7 loop structure of the β-domain of the VHL protein (pVHL), destabilizes the VHL-HIF-1αcomplex, and induces the truncation of pVHL. We speculate that the p.Q145X nonsense mutation leads to relatively obvious familial aggregation. This mutation causes the type I phenotype of VHL disease and is associated with a high risk of retinal and central nervous system (CNS) hemangioblastomas (HGBs) and visceral cysts, but a low risk of renal cell carcinoma. Moreover, within a VHL family, the average ages of first onset became younger in successive generations, and the CNS HGBs are more likely to occur in male patients.

A leucine residue in the L2 loop is critical for caspases 3 and 7 active site formation

A leucine residue in the L2 loop is critical for caspases 3 and 7 active site formation

Casitas B-Lineage Lymphoma RING Domain Inhibitors Protect Mice against High-Fat Diet-Induced Obesity and Insulin Resistance.

The casitas b-lineage lymphoma (c-Cbl) is an important adaptor protein with an intrinsic E3 ubiquitin ligase activity that interacts with E2 proteins such as UbCH7. c-Cbl plays a vital role in regulating receptor tyrosine kinase signaling. c-Cbl involves in whole-body energy homeostasis, which makes it a potential target for the treatment of type 2 diabetes and obesity. In the present study, we have designed two parental peptides and 55 modified peptides based on the structure of UbCH7 loop L1 and L2. Thirteen of the modified peptides showed increased inhibitory activity in a fluorescence polarization-based assay. In the in vivo proof of study principle, mice treated with peptides 10, 34, 49 and 51 were protected against high-fat diet-induced obesity and insulin resistant. These inhibitors may potentially lead to new therapeutic alternatives for obesity and type 2 diabetes.

Dynamic interplay of membrane-proximal POTRA domain and conserved loop L6 in Omp85 transporter FhaC.

Omp85 transporters mediate protein insertion into, or translocation across, membranes. They have a conserved architecture, with POTRA domains that interact with substrate proteins, a 16-stranded transmembrane β barrel, and an extracellular loop, L6, folded back in the barrel pore. Here using electrophysiology, in vivo biochemical approaches and electron paramagnetic resonance, we show that the L6 loop of the Omp85 transporter FhaC changes conformation and modulates channel opening. Those conformational changes involve breaking the conserved interaction between the tip of L6 and the inner β-barrel wall. The membrane-proximal POTRA domain also exchanges between several conformations, and the binding of FHA displaces this equilibrium. We further demonstrate a dynamic, physical communication between the POTRA domains and L6, which must take place via the β barrel. Our findings thus link all three essential components of Omp85 transporters and indicate that they operate in a concerted fashion in the transport cycle.

Structures of FolT in substrate-bound and substrate-released conformations reveal a gating mechanism for ECF transporters.

Energy-coupling factor (ECF) transporters are a new family of ABC transporters that consist of four subunits, two cytoplasmic ATPases EcfA and EcfA' and two transmembrane proteins namely EcfS for substrate-specific binding and EcfT for energy coupling. Here, we report the 3.2-Å resolution crystal structure of the EcfS protein of a folate ECF transporter from Enterococcus faecalis-EfFolT, a close homologue of FolT from Lactobacillus brevis-LbFolT. Structural and biochemical analyses reveal the residues constituting the folate-binding pocket and determining the substrate-binding specificity. Structural comparison of the folate-bound EfFolT with the folate-free LbFolT contained in the holotransporter complex discloses significant conformational change at the L1 loop, and reveals a gating mechanism of ECF transporters in which the L1 loop of EcfS acts as a gate in the substrate binding and release.

Rigidity Emerges during Antibody Evolution in Three Distinct Antibody Systems: Evidence from QSFR Analysis of Fab Fragments.

The effects of somatic mutations that transform polyspecific germline (GL) antibodies to affinity mature (AM) antibodies with monospecificity are compared among three GL-AM Fab pairs. In particular, changes in conformational flexibility are assessed using a Distance Constraint Model (DCM). We have previously established that the DCM can be robustly applied across a series of antibody fragments (VL to Fab), and subsequently, the DCM was combined with molecular dynamics (MD) simulations to similarly characterize five thermostabilizing scFv mutants. The DCM is an ensemble based statistical mechanical approach that accounts for enthalpy/entropy compensation due to network rigidity, which has been quite successful in elucidating conformational flexibility and Quantitative Stability/Flexibility Relationships (QSFR) in proteins. Applied to three disparate antibody systems changes in QSFR quantities indicate that the VH domain is typically rigidified, whereas the VL domain and CDR L2 loop become more flexible during affinity maturation. The increase in CDR H3 loop rigidity is consistent with other studies in the literature. The redistribution of conformational flexibility is largely controlled by nonspecific changes in the H-bond network, although certain Arg to Asp salt bridges create highly localized rigidity increases. Taken together, these results reveal an intricate flexibility/rigidity response that accompanies affinity maturation.

Conserved Omp85 lid-lock structure and substrate recognition in FhaC.

Omp85 proteins mediate translocation of polypeptide substrates across and into cellular membranes. They share a common architecture comprising substrate-interacting POTRA domains, a C-terminal 16-stranded β-barrel pore and two signature motifs located on the inner barrel wall and at the tip of the extended L6 loop. The observation of two distinct conformations of the L6 loop in the available Omp85 structures previously suggested a functional role of conformational changes in L6 in the Omp85 mechanism. Here we present a 2.5 Å resolution structure of a variant of the Omp85 secretion protein FhaC, in which the two signature motifs interact tightly and form the conserved ‘lid lock'. Reanalysis of previous structural data shows that L6 adopts the same, conserved resting state position in all available Omp85 structures. The FhaC variant structure further reveals a competitive mechanism for the regulation of substrate binding mediated by the linker to the N-terminal plug helix H1.

IR-spectroscopic characterization of an elongated OmpG mutant

OmpG is a nonselective, pH dependent outer membrane protein from Escherichia coli. It consists of 281 residues, forming a 14-stranded β-sheet structure. In this study, OmpG is extended by 38 amino acids to produce a 16-stranded β-barrel (OmpG-16S). The resulting protein is investigated by IR-spectroscopy. The secondary structure, pH-dependent opening/closing mechanism, buffer accessibility and thermal stability of OmpG-16S are compared to OmpG-WT. The results show that OmpG-16S is responsive to pH change as indicated by the Amide I band shift upon a switch from acidic to neutral pH. This spectral shift is consistent with that observed in OmpG-WT, which confirms the existence of structural differences consistent with the presence of the open or closed state. Secondary structure analysis after curve-fitting of Amide I band revealed that the additional residues do not fold into β-sheet; rather they are in the form of turns and unordered structure. In thermal stability experiments, OmpG-16S is found to be as stable as OmpG-WT. Additionally, H/D exchange experiments showed no difference in the exchange rate of OmpG-16S between the acidic and alkaline pH, suggesting that the loop L6 is no longer sufficient to block the pore entrance at acidic pH.

Identifying a potential receptor for the antibacterial peptide of sponge Axinella donnani endosymbiont

Marine sponges and their associated bacteria are rich sources of novel secondary metabolites with therapeutic usefulness. In our earlier work, we have identified a novel antibacterial peptide from the marine sponge Axinella donnani endosymbiotic bacteria. In this work, we have carried out a comparative genomic analysis and identified a set of 60 proteins as probable receptor which is common in all the strains. The analysis on binding substrate showed that β barrel assembly machinery (BamA) of the outer membrane protein 85 (omp85) superfamily is a potential receptor protein for the antibacterial peptide. It plays a central role in OMP biogenesis, especially in cell viability. Further, the triplet and quartet motifs RGF and YGDG, respectively in L6 loop are conserved over all the strains and these conserved residues interact with antibacterial peptide to inhibit the BamA function, which is essential for OMP biogenesis.

Structural Point Mutations of p53 Protein and Their Effects on the Zinc Coordination Complex

The p53 protein is an important transcription factor in the cell cycle. As such, mutations of this protein are found in at least 50% of all cancers, most of which are in the DNA‐binding domain containing residues 102‐292. This research is focusing on the molecular dynamics of p53 with point mutations that affect the structural stability of the protein, namely, R175H, R282W, G245S, and R249S. The system will be evaluated in reference to the Zn2+ cation that is tetrahedrally coordinated to Cys176, Cys238, Cys242, and His179. Changes in the stability of the coordination complex have been shown to have a greater effect on the stability of the overall protein, as it restricts the movement of the L2 and L3 loops. These systems and their changes are evaluated using molecular dynamic simulations with the AMBER12 suite, cpptraj, VMD and PyMOL. The objective of this study is to investigate how point mutations change the structural stability of the protein in relation to the zinc complex. Structural metrics including distance and surface measures shed light of the effects of these mutations on the dynamics of the protein. This informs future research initiatives for rational drug design.

Unique ATPase Site Architecture Triggers cis-Mediated Synchronized ATP Binding in Heptameric AAA+-ATPase Domain of Flagellar Regulatory Protein FlrC

Bacterial enhancer-binding proteins (bEBPs) oligomerize through AAA(+) domains and use ATP hydrolysis-driven energy to isomerize the RNA polymerase-σ(54) complex during transcriptional initiation. Here, we describe the first structure of the central AAA(+) domain of the flagellar regulatory protein FlrC (FlrC(C)), a bEBP that controls flagellar synthesis in Vibrio cholerae. Our results showed that FlrC(C) forms heptamer both in nucleotide (Nt)-free and -bound states without ATP-dependent subunit remodeling. Unlike the bEBPs such as NtrC1 or PspF, a novel cis-mediated "all or none" ATP binding occurs in the heptameric FlrC(C), because constriction at the ATPase site, caused by loop L3 and helix α7, restricts the proximity of the trans-protomer required for Nt binding. A unique "closed to open" movement of Walker A, assisted by trans-acting "Glu switch" Glu-286, facilitates ATP binding and hydrolysis. Fluorescence quenching and ATPase assays on FlrC(C) and mutants revealed that although Arg-349 of sensor II, positioned by trans-acting Glu-286 and Tyr-290, acts as a key residue to bind and hydrolyze ATP, Arg-319 of α7 anchors ribose and controls the rate of ATP hydrolysis by retarding the expulsion of ADP. Heptameric state of FlrC(C) is restored in solution even with the transition state mimicking ADP·AlF3. Structural results and pulldown assays indicated that L3 renders an in-built geometry to L1 and L2 causing σ(54)-FlrC(C) interaction independent of Nt binding. Collectively, our results underscore a novel mechanism of ATP binding and σ(54) interaction that strives to understand the transcriptional mechanism of the bEBPs, which probably interact directly with the RNA polymerase-σ(54) complex without DNA looping.

Characterization of the Readout Mechanism and the L1 Loop's Role in the p53 Tumor Suppressor Protein Binding Event via Molecular Dynamics Simulations

A primary target in cancer research, the p53 tumor suppressor protein, is a transcription activator playing an integral role in the regulation of the cell cycle. Through binding to DNA, the protein has the ability to “turn on” other repair proteins to mend damaged DNA, inhibit cellular division, and initiate apoptosis in cells that are damaged beyond repair. Most inactive p53 mutants contain changes primarily localized to the DNA binding domain (DBD). While intensive research has been conducted on different p53 mutants, previous studies have not yet focused on the capability for the system to exhibit direct and indirect readout among various genomic sites. Consequently, the L1 loop, a structural feature of the DBD, participates in binding to sequences exhibiting differences in the readout mechanism. This study aims to characterize the dynamic properties of the p53 binding event. Hydrogen bonding as direct readout will be ascertained from additive energy models and DNA deformation mechanics will be explored as indirect readout from molecular dynamics (MD) simulations. Additionally, this experiment illuminates the contribution of the L1 loop to the p53/DNA binding interface. Molecular models of p53 with DNA expected to have differing hydrogen bonding patterns are constructed and simulated using the Amber12 MD suite. Results from this study inform rational design of novel therapeutics to combat the changes that are hypothesized to cause over 50% of tumorous cancers.

Three‐dimensional Domain Swapping in the α Subunit of Tryptophan Synthase

Tryptophan synthase is an enzyme participating in the last two steps of tryptophan synthesis in plants, fungi and bacteria. It consists of two protein chains, α and β, encoded by trpA and trpB genes, that function as an αββα complex. Structural and functional features of tryptophan synthase have been extensively studied, explaining roles of individual residues in catalysis and allosteric regulation of the two active sites. TrpA has become a model system to investigate folding mechanisms of larger proteins. In 1969, Jackson and Yanofsky observed that typically monomeric TrpA forms a small population of dimers. The dimerization was postulated to occur through an exchange of structural elements of the monomeric chains – a phenomenon currently known as 3D domain swapping. Nevertheless, structural details of the dimeric form of TrpA protein remained unknown. We determined the crystal structure of TrpA that for the first time illustrates domain swapping in a TIM barrel fold. The exchanging fragment represents the N‐terminal section and includes H0‐S1‐H1‐S2 elements. The hinge region corresponds to loop L2 linking S2 with H2'. S2 and L2 carry the catalytic residues, Glu and Asp. As the S2 element is part of a swapped domain, architecture of the catalytic apparatus in α2 is regenerated from the two protein chains. The dimer interface corresponds to the α‐β interface in the tryptophan synthase complex, suggesting that the observed α2 dimer may not form a functional complex with the β subunit.This work was supported by the NIH grant GM094585 (MCSG) and the U.S. DOE, OBER, contract DE‐AC02‐06CH11357 (SBC).

Variable active site loop conformations accommodate the binding of macrocyclic largazole analogues to HDAC8.

The macrocyclic depsipeptide Largazole is a potent inhibitor of metal-dependent histone deacetylases (HDACs), some of which are drug targets for cancer chemotherapy. Indeed, Largazole partially resembles Romidepsin (FK228), a macrocyclic depsipeptide already approved for clinical use. Each inhibitor contains a pendant side chain thiol that coordinates to the active site Zn(2+) ion, as observed in the X-ray crystal structure of the HDAC8-Largazole complex [Cole, K. E., Dowling, D. P., Boone, M. A., Phillips, A. J., and Christianson, D. W. (2011) J. Am. Chem. Soc. 133, 12474]. Here, we report the X-ray crystal structures of HDAC8 complexed with three synthetic analogues of Largazole in which the depsipeptide ester is replaced with a rigid amide linkage. In two of these analogues, a six-membered pyridine ring is also substituted (with two different orientations) for the five-membered thiazole ring in the macrocycle skeleton. The side chain thiol group of each analogue coordinates to the active site Zn(2+) ion with nearly ideal geometry, thereby preserving the hallmark structural feature of inhibition by Largazole. Surprisingly, in comparison with the binding of Largazole, these analogues trigger alternative conformational changes in loops L1 and L2 flanking the active site. However, despite these structural differences, inhibitory potency is generally comparable to, or just moderately less than, the inhibitory potency of Largazole. Thus, this study reveals important new structure-affinity relationships for the binding of macrocyclic inhibitors to HDAC8.

Structure of a complex of uridine phosphorylase from Yersinia pseudotuberculosis with the modified bacteriostatic antibacterial drug determined by X-ray crystallography and computer analysis

Pseudotuberculosis and bubonic plague are acute infectious diseases caused by the bacteria Yersinia pseudotuberculosis and Yersinia pestis. These diseases are treated, in particular, with trimethoprim and its modified analogues. However, uridine phosphorylases (pyrimidine nucleoside phosphorylases) that are present in bacterial cells neutralize the action of trimethoprim and its modified analogues on the cells. In order to reveal the character of the interaction of the drug with bacterial uridine phosphorylase, the atomic structure of the unligated molecule of uridine-specific pyrimidine nucleoside phosphorylase from Yersinia pseudotuberculosis (YptUPh) was determined by X-ray diffraction at 1.7 A resolution with high reliability (R work = 16.2, R free = 19.4%; r.m.s.d. of bond lengths and bond angles are 0.006 A and 1.005°, respectively; DPI = 0.107 A). The atoms of the amino acid residues of the functionally important secondary-structure elements—the loop L9 and the helix H8—of the enzyme YptUPh were located. The three-dimensional structure of the complex of YptUPh with modified trimethoprim—referred to as 53I—was determined by the computer simulation. It was shown that 53I is a pseudosubstrate of uridine phosphorylases, and its pyrimidine-2,4-diamine group is located in the phosphate-binding site of the enzyme YptUPh.

Allosteric Effects of the Oncogenic RasQ61L Mutant on Raf-RBD

The Ras/Raf/MEK/ERK signal transduction pathway is a major regulator of cell proliferation activated by Ras-guanosine triphosphate (GTP). The oncogenic mutant RasQ61L is not able to hydrolyze GTP in the presence of Raf and thus is a constitutive activator of this mitogenic pathway. The Ras/Raf interaction is essential for the activation of the Raf kinase domain through a currently unknown mechanism. We present the crystal structures of the Ras-GppNHp/Raf-RBD and RasQ61L-GppNHp/Raf-RBD complexes, which, in combination with MD simulations, reveal differences in allosteric interactions leading from the Ras/Raf interface to the Ras calcium-binding site and to the remote Raf-RBD loop L4. In the presence of Raf, the RasQ61L mutant has a rigid switch II relative to the wild-type and increased flexibility at the interface with switch I, which propagates across Raf-RBD. We show that in addition to local perturbations on Ras, RasQ61L has substantial long-range effects on the Ras allosteric lobe and on Raf-RBD.

Characterization of the insertase BamA in three different membrane mimetics by solution NMR spectroscopy

The insertase BamA is the central protein of the Bam complex responsible for outer membrane protein biogenesis in Gram-negative bacteria. BamA features a 16-stranded transmembrane β-barrel and five periplasmic POTRA domains, with a total molecular weight of 88 kDa. Whereas the structure of BamA has recently been determined by X-ray crystallography, its functional mechanism is not well understood. This mechanism comprises the insertion of substrates from a dynamic, chaperone-bound state into the bacterial outer membrane, and NMR spectroscopy is thus a method of choice for its elucidation. Here, we report solution NMR studies of different BamA constructs in three different membrane mimetic systems: LDAO micelles, DMPC:DiC7PC bicelles and MSP1D1:DMPC nanodiscs. The impact of biochemical parameters on the spectral quality was investigated, including the total protein concentration and the detergent:protein ratio. The barrel of BamA is folded in micelles, bicelles and nanodiscs, but the N-terminal POTRA5 domain is flexibly unfolded in the absence of POTRA4. Measurements of backbone dynamics show that the variable insertion region of BamA, located in the extracellular lid loop L6, features high local flexibility. Our work establishes biochemical preparation schemes for BamA, which will serve as a platform for structural and functional studies of BamA and its role within the Bam complex by solution NMR spectroscopy.

Loop L1 governs the DNA-binding specificity and order for RecA-catalyzed reactions in homologous recombination and DNA repair.

In all organisms, RecA-family recombinases catalyze homologous joint formation in homologous genetic recombination, which is essential for genome stability and diversification. In homologous joint formation, ATP-bound RecA/Rad51-recombinases first bind single-stranded DNA at its primary site and then interact with double-stranded DNA at another site. The underlying reason and the regulatory mechanism for this conserved binding order remain unknown. A comparison of the loop L1 structures in a DNA-free RecA crystal that we originally determined and in the reported DNA-bound active RecA crystals suggested that the aspartate at position 161 in loop L1 in DNA-free RecA prevented double-stranded, but not single-stranded, DNA-binding to the primary site. This was confirmed by the effects of the Ala-replacement of Asp-161 (D161A), analyzed directly by gel-mobility shift assays and indirectly by DNA-dependent ATPase activity and SOS repressor cleavage. When RecA/Rad51-recombinases interact with double-stranded DNA before single-stranded DNA, homologous joint-formation is suppressed, likely by forming a dead-end product. We found that the D161A-replacement reduced this suppression, probably by allowing double-stranded DNA to bind preferentially and reversibly to the primary site. Thus, Asp-161 in the flexible loop L1 of wild-type RecA determines the preference for single-stranded DNA-binding to the primary site and regulates the DNA-binding order in RecA-catalyzed recombinase reactions.

Molecular Dynamics Approach in the Comparison of Wild-Type and Mutant Paraoxonase-1 Apoenzyme Form.

There is some evidence linking the mammalian paraoxonase-1 (PON1) loops (L1 and L2) to an increased flexibility and reactivity of its active site with potential substrates. The aim of this work is to study the structural, dynamical, and functional effects of the most flexible regions close to the active site and to determine the impact of mutations on the protein. For both models, wild-type (PON1wild) and PON1 mutant (PON1mut) models, the L1 loop and Q/R and L/M mutations were constructed using MODELLER software. Molecular dynamics simulations of 20 ns at 300 K on fully modeled PON1wild and PON1mut apoenzyme have been done. Detailed analyses of the root-mean-square deviation and fluctuations, H-bonding pattern, and torsion angles have been performed. The PON1wild results were then compared with those obtained for the PON1mut. Our results show that the active site in the wild-type structure is characterized by two distinct movements of opened and closed conformations of the L1 and L2 loops. The alternating and repetitive movement of loops at specific times is consistent with the presence of 11 defined hydrogen bonds. In the PON1mut, these open-closed movements are therefore totally influenced and repressed by the Q/R and L/M mutations. In fact, these mutations seem to impact the PON1mut active site by directly reducing the catalytic core flexibility, while maintaining a significant mobility of the switch regions delineated by the loops surrounding the active site. The impact of the studied mutations on structure and dynamics proprieties of the protein may subsequently contribute to the loss of both flexibility and activity of the PON1 enzyme.