Conformation and Trimer Association of the Transmembrane Domain of the Parainfluenza Virus Fusion Protein in Lipid Bilayers from Solid-State NMR: Insights into the Sequence Determinants of Trimer Structure and Fusion Activity

Enveloped viruses enter cells by using their fusion proteins to merge the virus lipid envelope and the cell membrane. While crystal structures of the water-soluble ectodomains of many viral fusion proteins have been determined, the structure and assembly of the C-terminal transmembrane domain (TMD) remains poorly understood. Here we use solid-state NMR to determine the backbone conformation and oligomeric structure of the TMD of the parainfluenza virus 5 fusion protein. 13C chemical shifts indicate that the central leucine-rich segment of the TMD is α-helical in POPC/cholesterol membranes and POPE membranes, while the Ile- and Val-rich termini shift to the β-strand conformation in the POPE membrane. Importantly, lipid mixing assays indicate that the TMD is more fusogenic in the POPE membrane than in the POPC/cholesterol membrane, indicating that the β-strand conformation is important for fusion by inducing membrane curvature. Incorporation of para-fluorinated Phe at three positions of the α-helical core allowed us to measure interhelical distances using 19F spin diffusion NMR. The data indicate that, at peptide:lipid molar ratios of ~ 1:15, the TMD forms a trimeric helical bundle with inter-helical distances of 8.2–8.4 Å for L493F and L504F and 10.5 Å for L500F. These data provide high-resolution evidence of trimer formation of a viral fusion protein TMD in phospholipid bilayers, and indicate that the parainfluenza virus 5 fusion protein TMD harbors two functions: the central α-helical core is the trimerization unit of the protein, while the two termini are responsible for inducing membrane curvature by transitioning to a β-sheet conformation. Keywords: magic-angle-spinning NMR; trimer formation; conformational plasticity; spin diffusionNational Institutes of Health (U.S.) (Grant GM066976

A new perspective on the analysis of helix-helix packing preferences in globular proteins

For many years it had been believed that steric compatibility of helix interfaces could be the source of the observed preference for particular angles between neighbouring helices as emerging from statistical analysis of protein databanks. Several elegant models describing how side chains on helices can interdigitate without steric clashes were able to account quite reasonably for the observed distributions. However, it was later recognized (Bowie, 1997 and Walther, 1998) that the ``bare'' measured angle distribution should be corrected to avoid statistical bias. Disappointingly, the rescaled distributions dramatically lost their similarity with theoretical predictions casting many doubts on the validity of the geometrical assumptions and models. In this report we elucidate a few points concerning the proper choice of the random reference distribution. In particular we show the existence of crucial corrections due to the correct implementation of the approach used to discriminate whether two helices are in contact or not and to measure their relative orientations. By using this new rescaling, the ``true'' packing angle preferences are well described, even more than with the original ``bare'' distribution, by regular packing models.Comment: 23 pages, 5 figure

Unmasking the Annexin I Interaction from the Structure of Apo-S100A11

AbstractS100A11 is a homodimeric EF-hand calcium binding protein that undergoes a calcium-induced conformational change and interacts with the phospholipid binding protein annexin I to coordinate membrane association. In this work, the solution structure of apo-S100A11 has been determined by NMR spectroscopy to uncover the details of its calcium-induced structural change. Apo-S100A11 forms a tight globular structure having a near antiparallel orientation of helices III and IV in calcium binding site II. Further, helices I and IV, and I and I′, form a more closed arrangement than observed in other apo-S100 proteins. This helix arrangement in apo-S100A11 partially buries residues in helices I (P3, E11, A15), III (V55, R58, M59), and IV (A86, C87, S90) and the linker (A45, F46), which are required for interaction with annexin I in the calcium-bound state. In apo-S100A11, this results in a “masked” binding surface that prevents annexin I binding but is uncovered upon calcium binding

De Novo Design of Copper Metallopeptides Capable of Electron Transfer: From Design to Function.

Biophysical characterization on de novo designed three-helical bundles is presented. The structure was determined to establish its physical integrity. Spectroscopic, electrochemical and photophysical studies were used to characterize designed redox-active copper sites. α3D, a de novo designed peptide that preassembles into a three-helix bundle fold, was functionalized with a triscysteine site to produce α3DIV. α3DIV structure was solved using Nuclear Magnetic Resonance (NMR). α3DIV comprised 1067 NMR restraints and 138 dihedral angles. The backbone of the 20 lowest energy structures has a root mean square deviation from the mean structure of 0.79 (0.16) Å, demonstrating a well-defined structure. The asymmetric 2HisCys(Met) copper electron transfer site, which is encompassed in the β-barrel fold of cupredoxins, was incorporated in α3D to examine whether the function and physical properties of cupredoxins can be recapitulated in an unrelated fold. This generated three designs: core, chelate and chelate-core constructs. Cu(II) binding to the core and chelate constructs displayed intense absorption bands between 380-400 nm (~2000 M−1 cm−1); the chelate-core construct showed two intense absorption bands at 401 (4429 M−1 cm−1) and 499 (2020 M−1 cm−1). X-ray absorption spectroscopy analysis on the Cu(I) adducts recapitulated the reduced state of cupredoxin proteins, producing short Cu-S(Cys) bonds at 2.16 – 2.23 Å. Overall, these results showed that the designed cupredoxin sites cannot enforce the structural constraints necessary for the appropriate Cu(II) chromophore, however the Cu(I) environment was retained. Moreover, the redox activity of the designed constructs was tested using electrochemical and photophysical methods. Electrochemical studies showed reduction potentials of +362 – +462 mV (vs. NHE), which are in the range of cupredoxins. Photophysical work revealed intermolecular ET activity with ruthenium(III)trisbipyridine produced first-order and bimolecular rate constants of 105 s−1 and 108 s−1 M−1, respectively. This work illustrates that the redox function of a native copper center in a β-barrel fold can be achieved in the α-helical framework of α3D. Further, the structure of α3DIV revealed a distorted triscysteine site, offering a model for proteins with thiol-rich ligands. Ultimately, this work provides a foundation for investigating long-range electron transfer reaction using de novo protein design.PHDChemistryUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/113318/1/plegaria_1.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/113318/2/plegaria_2.pd

Predicted Structures and Dynamics for Agonists and Antagonists Bound to Serotonin 5-HT2B and 5-HT2C Receptors

Subtype 2 serotonin (5-hydroxytryptamine, 5-HT) receptors are major drug targets for schizophrenia, feeding disorders, perception, depression, migraines, hypertension, anxiety, hallucinogens, and gastrointestinal dysfunctions.' We report here the predicted structure of 5-HT2B and 5-HT2C receptors bound to highly potent and selective 5-HT2B antagonist PRX-08066 3, (pKi: 30 nM), including the key binding residues [V103 (2.53), L132 (3.29), V190 (4.60), and L347 (6.58)] determining the selectivity of binding to 5-HT2B over 5-HT2A. We also report structures of the endogenous agonist (5 HT) and a HT2B selective antagonist 2 (1-methyl-1-1,6,7,8-tetrahydro-pyrrolo [2,3-g]quinoline-5-carboxylic acid pyridine-3-ylamide). We examine the dynamics for the agonist-and antagonist-bound HT2B receptors in explicit membrane and water finding dramatically different patterns of water migration into the NPxxY motif and the binding site that correlates with the stability of ionic locks in the D(E)RY region

An Equilibrium-Dependent Retroviral mRNA Switch Regulates Translational Recoding

Most retroviruses require translational recoding of a viral messenger RNA stop codon to maintain a precise ratio of structural (Gag) and enzymatic (Pol) proteins during virus assembly. Pol is expressed exclusively as a Gag–Pol fusion either by ribosomal frameshifting or by read-through of the gag stop codon. Both of these mechanisms occur infrequently and only affect 5–10% of translating ribosomes, allowing the virus to maintain the critical Gag to Gag–Pol ratio. Although it is understood that the frequency of the recoding event is regulated by cis RNA motifs, no mechanistic explanation is currently available for how the critical protein ratio is maintained. Here we present the NMR structure of the murine leukaemia virus recoding signal and show that a protonation-dependent switch occurs to induce the active conformation. The equilibrium is such that at physiological pH the active, read-through permissive conformation is populated at approximately 6%: a level that correlates with in vivo protein quantities. The RNA functions by a highly sensitive, chemo-mechanical coupling tuned to ensure an optimal read-through frequency. Similar observations for a frameshifting signal indicate that this novel equilibrium-based mechanism may have a general role in translational recoding.Molecular and Cellular Biolog

Structure and dynamics of a constitutively active neurotensin receptor

Many G protein-coupled receptors show constitutive activity, resulting in the production of a second messenger in the absence of an agonist; and naturally occurring constitutively active mutations in receptors have been implicated in diseases. To gain insight into mechanistic aspects of constitutive activity, we report here the 3.3 Å crystal structure of a constitutively active, agonist-bound neurotensin receptor (NTSR1) and molecular dynamics simulations of agonist-occupied and ligand-free receptor. Comparison with the structure of a NTSR1 variant that has little constitutive activity reveals uncoupling of the ligand-binding domain from conserved connector residues, that effect conformational changes during GPCR activation. Furthermore, molecular dynamics simulations show strong contacts between connector residue side chains and increased flexibility at the intracellular receptor face as features that coincide with robust signalling in cells. The loss of correlation between the binding pocket and conserved connector residues, combined with altered receptor dynamics, possibly explains the reduced neurotensin efficacy in the constitutively active NTSR1 and a facilitated initial engagement with G protein in the absence of agonist

Identification of Regions Responsible for the Open Conformation of S100A10 Using Chimaeric S100A11/S100A10 Proteins

S100A11 is a dimeric, EF-hand calcium-binding protein. Calcium binding to S100A11 results in a large conformational change that uncovers a broad hydrophobic surface used to interact with phospholipid-binding proteins (annexins A1 and A2), and facilitate membrane vesiculation events. In contrast to other S100 proteins, S100A10 is unable to bind calcium due to deletion and substitution of calcium-ligating residues. Despite this, calcium-free S100A10 assumes an “open” conformation that is very similar to S100A11 in its calcium-bound state (Ca2+-S100A11). To understand how S100A10 is able to adopt an open conformation in the absence of calcium, seven chimeric proteins were constructed where regions from calcium-binding sites I and II, and helices II-IV in S100A11 were replaced with the corresponding regions of S100A10. The chimeric proteins having substitutions in calcium-binding site II displayed increased hydrophobic surface exposure as assessed by ANS fluorescence and phenyl Sepharose binding in the absence of calcium. This response is similar to that observed for Ca2+-S100A11 and calcium-free S100A10. Further, this substitution resulted in calcium-insensitive binding to annexin A2 for one chimeric protein. The results indicate that residues within site II are important in stabilizing the open conformation of S100A10 and presentation of its target-binding site. In contrast, S100A11 chimeric proteins with helical substitutions displayed poorer hydrophobic surface exposure and consequently, unobservable annexin A2 binding. This work represents a first attempt to systematically understand the molecular basis for the calcium-insensitive open conformation of S100A10

Transmembrane Domain Structure and Function in the Erythropoietin Receptor

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