Vladimir Ladizhansky

Structural Polymorphism and Multifunctionality of Myelin Basic Protein

Central nervous system myelin is a dynamic entity arising from membrane processes extended from oligodendrocytes, which form a tightly wrapped multilamellar structure around neurons enabling rapid and efficient signal propagation. The gene of oligodendrocyte lineage (golli) gives rise to a variety of developmentally regulated splice isoforms of myelin basic protein (MBP), denoted golli for early forms and classic for later ones. In mature myelin, the predominant splice isoform of classic MBP is 18.5 kDa; its central role is to maintain the structural integrity of the myelin sheath, by holding together the apposing cytoplasmic leaflets of the oligodendrocyte membrane in a tight, spiral, multilamellar arrangement. This proteins extreme physicochemical properties, net charge of +19 at neutral pH, low proportion of hydrophobic residues, alternating regions of predicted intrinsic disorder and order, induced folding upon association with membranes and other proteins, and diversification via combinatorial post-translational modifications, define not only its role as a molecular Velcro in compact myelin, but as a multifunctional hub that may also bind to a number of other proteins and small molecule ligands in myelinating oligodendrocytes. In particular, MBP may link the underlying cytoskeleton and proteins containing SH3 domains to the membrane, allowing it to transduce transmembrane signals to the cytosol. These associations are facilitated by MBP being an intrinsically disordered protein, creating a large effective protein surface, and by the formation of transient and/or induced ordered secondary structure elements for molecular recognition. These processes can be modulated by a molecular barcode of numerous post-translational modifications and interactions with proteins such as calmodulin. In the human demyelinating disease multiple sclerosis, an aberrant pattern of modifications may contribute to demyelination and confound inherent attempts at repair. The conformational dynamics of the various isoforms and modified variants of MBP and their interactions with other proteins potentially allow them to participate in events coupling extracellular signals to cytoskeletal organization during myelination or remyelination. Various biophysical and cell biological approaches are beginning to elucidate these properties of MBP and are leading to a new understanding of the role of this protein as a linker and/or hub in structural and signaling networks in oligodendrocytes and myelin.

Solid-state NMR spectroscopy structure determination of a lipid-embedded heptahelical membrane protein

Determination of structure of integral membrane proteins, especially in their native environment, is a formidable challenge in structural biology. Here we demonstrate that magic angle spinning solid-state NMR spectroscopy can be used to determine structures of membrane proteins reconstituted in synthetic lipids, an environment similar to the natural membrane. We combined a large number of experimentally determined interatomic distances and local torsional restraints to solve the structure of an oligomeric membrane protein of common seven-helical fold, Anabaena sensory rhodopsin (ASR). We determined the atomic resolution detail of the oligomerization interface of the ASR trimer, and the arrangement of helices, side chains and the retinal cofactor in the monomer.

Proton-Detected Solid-State NMR Reveals Intramembrane Polar Networks in a Seven-Helical Transmembrane Protein Proteorhodopsin

We used high-resolution proton-detected multidimensional NMR to study the solvent-exposed parts of a seven-helical integral membrane proton pump, proteorhodopsin (PR). PR samples were prepared by growing the apoprotein on fully deuterated medium and reintroducing protons to solvent-accessible sites through exchange with protonated buffer. This preparation leads to NMR spectra with proton resolution down to ca. 0.2 ppm at fast spinning (28 kHz) in a protein back-exchanged at a level of 40%. Novel three-dimensional proton-detected chemical shift correlation spectroscopy allowed for the identification and resonance assignment of the solvent-exposed parts of the protein. Most of the observed residues are located at the membrane interface, but there are notable exceptions, particularly in helix G, where most of the residues are susceptible to H/D exchange. This helix contains Schiff base-forming Lys231, and many conserved polar residues in the extracellular half, such as Asn220, Tyr223, Asn224, Asp227, and Asn230. We proposed earlier that high mobility of the F-G loop may transiently expose a hydrophilic cavity in the extracellular half of the protein, similar to the one found in xanthorhodopsin. Solvent accessibility of helix G is in line with this hypothesis, implying that such a cavity may be a part of the proton-conducting pathway lined by this helix.

Solid-state NMR study of proteorhodopsin in the lipid environment: secondary structure and dynamics.

Proteorhodopsins are typical retinal-binding light-driven proton pumps of heptahelical architecture widely distributed in marine and freshwater bacteria. Recently, we have shown that green proteorhodopsin (GPR) can be prepared in a lipid-bound state that gives well-resolved magic angle spinning (MAS) NMR spectra in samples with different patterns of reverse labelling. Here, we present 3D and 4D sequential chemical shift assignments identified through experiments conducted on a uniformly (13)C,(15)N-labelled sample. These experiments provided the assignments for 153 residues, with a particularly high density in the transmembrane regions ( approximately 74% of residues). The extent of assignments permitted a detailed examination of the secondary structure and dynamics in GPR. In particular, we present experimental evidence of mobility of the proteins termini and of the A-B, C-D, and F-G loops, the latter being possibly coupled to the GPR ion-transporting function.

Paramagnetic relaxation enhancement reveals oligomerization interface of a membrane protein.

Protein-protein interactions play critical roles in cellular function and oligomerization of membrane proteins is a commonly observed phenomenon. Determining the oligomerization state and defining the intermolecular interface in the bilayer is generally a difficult task. Here, we use site-specific spin labeling to demonstrate that relaxation enhancements induced by covalently attached paramagnetic tag can provide distance restraints defining the intermonomer interface in oligomers formed by a seven-helical transmembrane protein Anabaena Sensory Rhodopsin (ASR). We combine these measurements with visible CD spectroscopy and cross-linking experiments to demonstrate that ASR forms tight trimers in both detergents and lipids.

Homonuclear dipolar recoupling techniques for structure determination in uniformly 13C-labeled proteins

In solid-state NMR magic angle spinning is often used to remove line broadening associated with anisotropic interactions, such as chemical shift anisotropy and dipolar couplings. Dipolar recoupling refers to sequences of pulses designed to reintroduce dipolar interactions that are otherwise averaged by magic angle spinning. One of the key applications of homonuclear (and heteronuclear) dipolar recoupling is for the purpose of protein structure determination. Recoupling experiments, originally designed for applications in spin-pair labeled samples, have been revised in recent years for applications in samples with extensive or uniform incorporation of isotopic labels. In these samples multiple internuclear distances can in principle be probed simultaneously, but the dipolar truncation effects (i.e. attenuation of the effects of weak couplings by strong ones) circumvent such measurements. In this article we review some of the recent developments in homonuclear recoupling methods that allow overcoming this problem.

Recent advances in magic angle spinning solid state NMR of membrane proteins

Membrane proteins mediate many critical functions in cells. Determining their three-dimensional structures in the native lipid environment has been one of the main objectives in structural biology. There are two major NMR methodologies that allow this objective to be accomplished. Oriented sample NMR, which can be applied to membrane proteins that are uniformly aligned in the magnetic field, has been successful in determining the backbone structures of a handful of membrane proteins. Owing to methodological and technological developments, Magic Angle Spinning (MAS) solid-state NMR (ssNMR) spectroscopy has emerged as another major technique for the complete characterization of the structure and dynamics of membrane proteins. First developed on peptides and small microcrystalline proteins, MAS ssNMR has recently been successfully applied to large membrane proteins. In this review we describe recent progress in MAS ssNMR methodologies, which are now available for studies of membrane protein structure determination, and outline a few examples, which highlight the broad capability of ssNMR spectroscopy.

Conformational dynamics of a seven transmembrane helical protein Anabaena Sensory Rhodopsin probed by solid-state NMR.

The ability to detect and characterize molecular motions represents one of the unique strengths of nuclear magnetic resonance (NMR) spectroscopy. In this study, we report solid-state NMR site-specific measurements of the dipolar order parameters and (15)N rotating frame spin-lattice (R1ρ) relaxation rates in a seven transmembrane helical protein Anabaena Sensory Rhodopsin reconstituted in lipids. The magnitudes of the observed order parameters indicate that both the well-defined transmembrane regions and the less structured intramembrane loops undergo restricted submicrosecond time scale motions. In contrast, the R1ρ rates, which were measured under fast magic angle spinning conditions, vary by an order of magnitude between the TM and exposed regions and suggest the presence of intermediate time scale motions. Using a simple model, which assumes a single exponential autocorrelation function, we estimated the time scales of dominant stochastic motions to be on the order of low tens of nanoseconds for most residues within the TM helices and tens to hundreds of nanoseconds for the extracellular B-C and F-G loops. These relatively slow time scales could be attributed to collective anisotropic motions. We used the 3D Gaussian axial fluctuations model to estimate amplitudes, directions, and time scales of overall motions for helices and the extracellular B-C and F-G loops. Within this model, the TM helices A,B,C,D,E,F undergo rigid body motions on a time scale of tens of nanoseconds, while the time scale for the seventh helix G approaches 100 ns. Similar time scales of roughly 100-200 ns are estimated for the B-C and F-G loops.

Solid-State NMR Spectroscopy of Membrane-Associated Myelin Basic Protein—Conformation and Dynamics of an Immunodominant Epitope

Myelin basic protein (MBP) maintains the tight multilamellar compaction of the myelin sheath in the central nervous system through peripheral binding of adjacent lipid bilayers of oligodendrocytes. Myelin instability in multiple sclerosis (MS) is associated with the loss of positive charge in MBP as a result of posttranslational enzymatic deimination. A highly-conserved central membrane-binding fragment (murine N81-PVVHFFKNIVTPRTPPP-S99, identical to human N83-S101) represents a primary immunodominant epitope in MS. Previous low-resolution electron paramagnetic resonance measurements on the V83-T92 fragment, with Cys-mutations and spin-labeling that scanned the epitope, were consistent with it being a membrane-associated amphipathic alpha-helix. Pseudodeimination at several sites throughout the protein, all distal to the central segment, disrupted the alpha-helix at its amino-terminus and exposed it to proteases, representing a potential mechanism in the autoimmune pathogenesis of MS. Here, we have used magic-angle spinning solid-state NMR spectroscopy to characterize more precisely the molecular conformation and dynamics of this central immunodominant epitope of MBP in a lipid milieu, without Cys-substitution. Our solid-state NMR measurements have revealed that the alpha-helix present within the immunodominant epitope is shorter than originally modeled, and is independent of the pseudodeimination, highlighting the importance of the local hydrophobic effects in helix formation and stability. The main effect of pseudodeimination is to cause the cytoplasmic exposure of the fragment, potentially making it more accessible to proteolysis. These results are the first, to our knowledge, to provide atomic-level detail of a membrane-anchoring segment of MBP, and direct evidence of decreased MBP-membrane interaction after posttranslational modification.

Induced Secondary Structure and Polymorphism in an Intrinsically Disordered Structural Linker of the CNS: Solid-State NMR and FTIR Spectroscopy of Myelin Basic Protein Bound to Actin

The 18.5 kDa isoform of myelin basic protein (MBP) is a peripheral membrane protein that maintains the structural integrity of the myelin sheath of the central nervous system by conjoining the cytoplasmic leaflets of oligodendrocytes and by linking the myelin membrane to the underlying cytoskeleton whose assembly it strongly promotes. It is a multifunctional, intrinsically disordered protein that behaves primarily as a structural stabilizer, but with elements of a transient or induced secondary structure that represent binding sites for calmodulin or SH3-domain-containing proteins, inter alia. In this study we used solid-state NMR (SSNMR) and Fourier transform infrared (FTIR) spectroscopy to study the conformation of 18.5 kDa MBP in association with actin microfilaments and bundles. FTIR spectroscopy of fully (13)C,(15)N-labeled MBP complexed with unlabeled F-actin showed induced folding of both protein partners, viz., some increase in beta-sheet content in actin, and increases in both alpha-helix and beta-sheet content in MBP, albeit with considerable extended structure remaining. Solid-state NMR spectroscopy revealed that MBP in MBP-actin assemblies is structurally heterogeneous but gains ordered secondary structure elements (both alpha-helical and beta-sheet), particularly in the terminal fragments and in a central immunodominant epitope. The overall conformational polymorphism of MBP is consistent with its in vivo roles as both a linker (membranes and cytoskeleton) and a putative signaling hub.