Tyler Reddy

Myf5-/- :MyoD-/- amyogenic fetuses reveal the importance of early contraction and static loading by striated muscle in mouse skeletogenesis.

The mechanical loading of striated muscle is thought to play an important role in shaping bones and joints. Here, we examine skeletogenesis in late embryogenesis (embryonic day 18.5) in Myf5−/−:MyoD−/− fetuses completely lacking striated muscle. The phenotype includes enlarged and fused cervical vertebrae and postural anomalies, some viscerocranial anomalies, long bone truncation and fusion, absent deltoid tuberosity of the humerus, scapular and clavicular hypoplasia, cleft palate, and cleft sternum. In contrast, neurocranial bone development was essentially normal. While the magnitude of individual effects varied throughout the skeletal system, the results are consistent with skeletal development depending on functional muscles. Novel abnormalities in the amyogenic fetuses relative to less severely paralyzed phenotypes extend our understanding of skeletogenic dependence on embryonic muscle contraction and static loading.

Structural insight into G-protein coupled receptor binding by apelin.

Apelin peptides are the cognate ligands for the G-protein coupled receptor APJ, with functions in the cardiovascular and central nervous systems, in glucose metabolism and as a human immunodeficiency virus (HIV-1) coreceptor. Apelin is found in 13-36 residue forms in vivo. The structures of five isoforms of apelin at physiological versus low (5-6 degrees C) temperature are compared here using circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopy, demonstrating increased structure at low temperature. Far-ultraviolet (UV) CD spectra are predominantly random coil for apelin isoforms, but are convoluted by unusual bands from the C-terminal phenylalanine side chain. These bands, assigned using F13A-apelin-13, are accentuated at 5 degrees C and imply conformational restriction. At 35 degrees C, the R6-L9 region of apelin-17 is well structured, consistent with previous mutagenesis results showing necessity of this segment for apelin-APJ binding and activation. At 5 degrees C, R6-L9 retains its structuring while the functionally critical C-terminal G13-F17 region also becomes highly structured. Type IV beta-turns and some polyproline-II structure alongside F17 side chain motional reduction correlate well with CD spectral properties. Cis-trans peptide bond isomerization at P14 and P16 produces two sequentially assignable conformers (both trans:both cis approximately 4:1) alongside less populated conformers. Chemical shift assignment of apelin-12, -13 and pyroglutamate-apelin-13 implies highly similar structuring and the same isomerization at the C-terminus. Based on the apelin-17 structure, a two-step binding and activation mechanism is hypothesized.

Nothing to Sneeze At: A Dynamic and Integrative Computational Model of an Influenza A Virion

Summary The influenza virus is surrounded by an envelope composed of a lipid bilayer and integral membrane proteins. Understanding the structural dynamics of the membrane envelope provides biophysical insights into aspects of viral function, such as the wide-ranging survival times of the virion in different environments. We have combined experimental data from X-ray crystallography, nuclear magnetic resonance spectroscopy, cryo-electron microscopy, and lipidomics to build a model of the intact influenza A virion. This is the basis of microsecond-scale coarse-grained molecular dynamics simulations of the virion, providing simulations at different temperatures and with varying lipid compositions. The presence of the Forssman glycolipid alters a number of biophysical properties of the virion, resulting in reduced mobility of bilayer lipid and protein species. Reduced mobility in the virion membrane may confer physical robustness to changes in environmental conditions. Our simulations indicate that viral spike proteins do not aggregate and thus are competent for multivalent immunoglobulin G interactions.

The Role of the Membrane in the Structure and Biophysical Robustness of the Dengue Virion Envelope

Summary The dengue virion is surrounded by an envelope of membrane proteins surrounding a lipid bilayer. We have combined the cryoelectron microscopy structures of the membrane proteins (PDB: 3J27) with a lipid bilayer whose composition is based on lipidomics data for insect cell membranes, to obtain a near-atomic resolution computational model of the envelope of the dengue virion. A coarse-grained molecular dynamics simulation on the microsecond timescale enables analysis of key biophysical properties of the dengue outer envelope. Properties analyzed include area per lipid values (for a spherical virion with a mixed lipid composition), bilayer thickness, and lipid diffusion coefficients. Despite the absence of cholesterol from the lipid bilayer, the virion exhibits biophysical robustness (slow lipid diffusion alongside stable bilayer thickness, virion diameter, and shape) that matches the cholesterol-rich membrane of influenza A, with similarly anomalous diffusion of lipids. Biophysical robustness of the envelope may confer resilience to environmental perturbations.

Structural features of the apelin receptor N-terminal tail and first transmembrane segment implicated in ligand binding and receptor trafficking.

G-protein coupled receptors (GPCRs) comprise a large family of membrane proteins with rich functional diversity. Signaling through the apelin receptor (AR or APJ) influences the cardiovascular system, central nervous system and glucose regulation. Pathophysiological involvement of apelin has been shown in atherosclerosis, cancer, human immunodeficiency virus-1 (HIV-1) infection and obesity. Here, we present the high-resolution nuclear magnetic resonance (NMR) spectroscopy-based structure of the N-terminus and first transmembrane (TM) segment of AR (residues 1-55, AR55) in dodecylphosphocholine micelles. AR55 consists of two disrupted helices, spanning residues D14-K25 and A29-R55(1.59). Molecular dynamics (MD) simulations of AR built from a hybrid of experimental NMR and homology model-based restraints allowed validation of the AR55 structure in the context of the full-length receptor in a hydrated bilayer. AR55 structural features were functionally probed using mutagenesis in full-length AR through monitoring of apelin-induced extracellular signal-regulated kinase (ERK) phosphorylation in transiently transfected human embryonic kidney (HEK) 293A cells. Residues E20 and D23 form an extracellular anionic face and interact with lipid headgroups during MD simulations in the absence of ligand, producing an ideal binding site for a cationic apelin ligand proximal to the membrane-water interface, lending credence to membrane-catalyzed apelin-AR binding. In the TM region of AR55, N46(1.50) is central to a disruption in helical character. G42(1.46), G45(1.49) and N46(1.50), which are all involved in the TM helical disruption, are essential for proper trafficking of AR. In summary, we introduce a new correlative NMR spectroscopy and computational biochemistry methodology and demonstrate its utility in providing some of the first high-resolution structural information for a peptide-activated GPCR TM domain.

Multifaceted substrate capture scheme of a rhomboid protease.

Rhomboid proteases are integral membrane serine proteases that catalyze peptide bond hydrolysis in biological membranes. Little is currently known about the interaction of enzyme and substrate. Coarse-grained molecular dynamics simulations in hydrated lipid bilayers are employed herein to study the interaction of the E. coli rhomboid protease GlpG (ecGlpG) with the transmembrane domain (TMD) of the substrate Spitz. Spitz does not associate with ecGlpG exclusively at the putative substrate gate near TMD 5. Instead, there are six prominent and stable interaction sites, including one between TMDs 1 and 3, with the closest enzyme-substrate proximity occurring at the ends of helical TMDs or in loops. Bilayer thinning is observed proximal to ecGlpG, but there is no evidence of additional thinning of the bilayer upon interaction with substrate. We suggest that the initial interaction between enzyme and substrate, or substrate capture event, is not limited to a single site on the enzyme, and may be driven by juxtamembrane electrostatic interactions. The findings are of additional interest because catalytically inactive rhomboids (iRhoms) are now known to interact with the substrates of their catalytically active counterparts and to antagonize the enzyme-driven pathways.

Computational virology: From the inside out.

Viruses typically pack their genetic material within a protein capsid. Enveloped viruses also have an outer membrane made up of a lipid bilayer and membrane-spanning glycoproteins. X-ray diffraction and cryoelectron microscopy provide high resolution static views of viral structure. Molecular dynamics (MD) simulations may be used to provide dynamic insights into the structures of viruses and their components. There have been a number of simulations of viral capsids and (in some cases) of the inner core of RNA or DNA packaged within them. These simulations have generally focussed on the structural integrity and stability of the capsid and/or on the influence of the nucleic acid core on capsid stability. More recently there have been a number of simulation studies of enveloped viruses, including HIV-1, influenza A, and dengue virus. These have addressed the dynamic behaviour of the capsid, the matrix, and/or of the outer envelope. Analysis of the dynamics of the lipid bilayer components of the envelopes of influenza A and of dengue virus reveals a degree of biophysical robustness, which may contribute to the stability of virus particles in different environments. Significant computational challenges need to be addressed to aid simulation of complex viruses and their membranes, including the need to integrate structural data from a range of sources to enable us to move towards simulations of intact virions. This article is part of a Special Issue entitled: Membrane Proteins edited by J.C. Gumbart and Sergei Noskov.

Primary and Secondary Dimer Interfaces of the Fibroblast Growth Factor Receptor 3 Transmembrane Domain: Characterization via Multiscale Molecular Dynamics Simulations

Receptor tyrosine kinases are single-pass membrane proteins that form dimers within the membrane. The interactions of their transmembrane domains (TMDs) play a key role in dimerization and signaling. Fibroblast growth factor receptor 3 (FGFR3) is of interest as a G380R mutation in its TMD is the underlying cause of ~99% of the cases of achondroplasia, the most common form of human dwarfism. The structural consequences of this mutation remain uncertain: the mutation shifts the position of the TMD relative to the lipid bilayer but does not alter the association free energy. We have combined coarse-grained and all-atom molecular dynamics simulations to study the dimerization of wild-type, heterodimer, and mutant FGFR3 TMDs. The simulations reveal that the helices pack together in the dimer to form a flexible interface. The primary packing mode is mediated by a Gx3G motif. There is also a secondary dimer interface that is more highly populated in heterodimer and mutant configurations that may feature in the molecular mechanism of pathology. Both coarse-grained and atomistic simulations reveal a significant shift of the G380R mutant dimer TMD relative to the bilayer to allow interactions of the arginine side chain with lipid headgroup phosphates.

Correlating structure, dynamics, and function in transmembrane segment VII of the Na+/H+exchanger isoform 1

We place (15)N nuclear magnetic resonance relaxation analysis and functional mutagenesis studies in the context of our previous structural and mutagenesis work to correlate structure, dynamics and function for the seventh transmembrane segment of the human Na(+)/H(+) exchanger isoform 1. Although G261-S263 was previously identified as an interruption point in the helical structure of this isolated transmembrane peptide in dodecylphosphocholine micelles, and rapid conformational exchange was implicated in the NOE measurements, the six (15)N labelled residues examined in this study all have similar dynamics on the ps-ns time scale. A mathematical model incorporating chemical exchange is the best fit for residues G261, L264, and A268. This implies that a segment of residues from G261 to A268 samples different conformations on the mus-ms time scale. Chemical exchange on an intermediate time scale is consistent with an alternating-access cycle where E262 is bent away from the cytosol during proton translocation by the exchanger. The functional importance of chemical exchange at G261-A268 is corroborated by the abrogated activity of the full-length exchanger with the bulky and restricting Ile substitutions F260I, G261I, E262I, S263I, and A268I.

Interpretation of biomolecular NMR spin relaxation parameters.

Biomolecular nuclear magnetic resonance (NMR) spin relaxation experiments provide exquisite information on the picosecond to nanosecond timescale motions of bond vectors. Spin-lattice (T1) and spin-spin (T2) relaxation times and the steady-state nuclear Overhauser effect (NOE) are the first set of parameters extracted from typical 15N or 13C NMR relaxation experiments. Therefore, verifying that T1, T2, and NOE are consistent with theoretical predictions is an important step before carrying out the more detailed model-free and reduced spectral density mapping analyses commonly employed. In this mini-review, we discuss the essential motional parameters used to describe biomolecular dynamics in the context of a variety of examples of folded and intrinsically disordered proteins and peptides in aqueous and membrane mimetic environments. Estimates of these parameters can be used as input for an online interface, introduced herein, allowing plotting of trends of T1, T2, and NOE with magnetic field strength. The plots may serve as a first-check to the spectroscopist preparing to embark on a detailed NMR relaxation analysis.