Ez, a Depth-dependent Potential for Assessing the Energies of Insertion of Amino Acid Side-chains into Membranes: Derivation and Applications to Determining the Orientation of Transmembrane and Interfacial Helices

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Phospholamban (PLN) is an essential regulator of cardiac muscle contractility. The homopentameric assembly of PLN is the reservoir for active monomers that, upon deoligomerization form 1:1 complexes with the sarco(endo)plasmic reticulum Ca 2؉ -ATPase (SERCA), thus modulating the rate of calcium uptake. In lipid bilayers and micelles, monomeric PLN exists in equilibrium between a bent (or resting) T state and a more dynamic (or active) R state. Here, we report the high-resolution structure and topology of the T state of a monomeric PLN mutant in lipid bilayers, using a hybrid of solution and solid-state NMR restraints together with molecular dynamics simulations in explicit lipid environments. Unlike the previous structural ensemble determined in micelles, this approach gives a complete picture of the PLN monomer structure in a lipid bilayer. This hybrid ensemble exemplifies the tilt, rotation, and depth of membrane insertion, revealing the interaction with the lipids for all protein domains. The N-terminal amphipathic helical domain Ia (residues 1-16) rests on the surface of the lipid membrane with the hydrophobic face of domain Ia embedded in the membrane bilayer interior. The helix comprised of domain Ib (residues 23-30) and transmembrane domain II (residues 31-52) traverses the bilayer with a tilt angle of Ϸ24°. The specific interactions between PLN and lipid membranes may represent an additional regulatory element of its inhibitory function. We propose this hybrid method for the simultaneous determination of structure and topology for membrane proteins with compact folds or proteins whose spatial arrangement is dictated by their specific interactions with lipid bilayers.hybrid method ͉ membrane proteins ͉ oriented solid-state NMR ͉ molecular modeling ͉ PISEMA S tructure and topology are central to membrane protein function (1). Recently determined high-resolution structures reveal the compact folds for several membrane proteins, such as electron and proton-conducting proteins involved in photosynthesis and respiration (http://blanco.biomol.uci.edu/ Membrane Proteins xtal.html). However, a significant population of membrane proteins does not possess a compact tertiary fold, but has its fold space defined through interactions of secondary structure elements (helices, turns, and loops) with the lipid membrane, i.e., the topology (1). This is the case for phospholamban (PLN), a mammalian protein that is essential in the regulation of cardiac muscle contractility (2), and that has recently become a major target for gene therapy to ameliorate cardiomyopathies (3, 4). PLN is located in the sarco(endo)plasmic reticulum (SR) of cardiac myocytes, inhibiting the SR Ca 2ϩ -ATPase (SERCA) by shifting its relative Ca 2ϩ affinity (5). In vitro and in vivo experiments have shown PLN to exist as a homopentamer that deoligomerizes into active monomers that bind SERCA in a 1:1 molar ratio (6). The monomeric form of PLN exists in equilibrium between a dynamically disordered R state and a more restricted T state (7,8) and has 3 ...

Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

Structure and topology of monomeric phospholamban in lipid membranes determined by a hybrid solution and solid-state NMR approach

Traaseth

Shi²,

Verardi

et al. 2009

157

194

“…Future work is necessary to refine, verify, and expand the scoring functions. For example, a membrane model, such as a depth-dependent potential (32) or an implicit solvent (33), is likely to improve the predictions and any correlation between the computational score and the A B C thermodynamic stability. Nevertheless, CATM appears to capture the essence of GAS right motifs already in the current form, and therefore the method is already applicable to the rapid prediction of unknown structures.…”

Section: Discussionmentioning

confidence: 99%

A frequent, GxxxG-mediated, transmembrane association motif is optimized for the formation of interhelical Cα–H hydrogen bonds

Mueller

Subramaniam

Senes

2014

Self Cite

143

Carbon hydrogen bonds between Cα-H donors and carbonyl acceptors are frequently observed between transmembrane helices (Cα-H···O=C). Networks of these interactions occur often at helix−helix interfaces mediated by GxxxG and similar patterns. Cα-H hydrogen bonds have been hypothesized to be important in membrane protein folding and association, but evidence that they are major determinants of helix association is still lacking. Here we present a comprehensive geometric analysis of homodimeric helices that demonstrates the existence of a single region in conformational space with high propensity for Cα-H···O=C hydrogen bond formation. This region corresponds to the most frequent motif for parallel dimers, GAS right , whose best-known example is glycophorin A. The finding suggests a causal link between the high frequency of occurrence of GAS right and its propensity for carbon hydrogen bond formation. Investigation of the sequence dependency of the motif determined that Gly residues are required at specific positions where only Gly can act as a donor with its "side chain" Hα. Gly also reduces the steric barrier for nonGly amino acids at other positions to act as Cα donors, promoting the formation of cooperative hydrogen bonding networks. These findings offer a structural rationale for the occurrence of GxxxG patterns at the GAS right interface. The analysis identified the conformational space and the sequence requirement of Cα-H···O=C mediated motifs; we took advantage of these results to develop a structural prediction method. The resulting program, CATM, predicts ab initio the known high-resolution structures of homodimeric GAS right motifs at near-atomic level.interaction motifs | protein prediction

“…The lipid bilayer becomes increasingly hydrophobic at greater depths within the membrane (31), and this environmental anisotropy is reflected in the amino acid composition of the barrel surface. Aliphatic side chains are prevalent toward the center of the membrane, and aromatic side chains are common in the lipid head group regions, where they encircle the barrel in external-and periplasmic-side girdles (32).Recently we developed E z β, a membrane depth-dependent, residue-level potential calculated from an ensemble of experimentally determined outer membrane protein structures (33,34). E z β can be used to estimate energetics of membrane insertion to predict transmembrane protein orientation within the bilayer, and to detect oligomerization sites on β-barrel surfaces (34).…”

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confidence: 99%

“…Recently we developed E z β, a membrane depth-dependent, residue-level potential calculated from an ensemble of experimentally determined outer membrane protein structures (33,34). E z β can be used to estimate energetics of membrane insertion to predict transmembrane protein orientation within the bilayer, and to detect oligomerization sites on β-barrel surfaces (34).…”

mentioning

confidence: 99%

Computational redesign of the lipid-facing surface of the outer membrane protein OmpA

Stapleton

Whitehead

Nanda

2015

Self Cite

Advances in computational design methods have made possible extensive engineering of soluble proteins, but designed β-barrel membrane proteins await improvements in our understanding of the sequence determinants of folding and stability. A subset of the amino acid residues of membrane proteins interact with the cell membrane, and the design rules that govern this lipid-facing surface are poorly understood. We applied a residue-level depth potential for β-barrel membrane proteins to the complete redesign of the lipid-facing surface of Escherichia coli OmpA. Initial designs failed to fold correctly, but reversion of a small number of mutations indicated by backcross experiments yielded designs with substitutions to up to 60% of the surface that did support folding and membrane insertion. T he β-barrel membrane proteins comprise one of the two structural classes of integral membrane proteins. They are found within the outer membranes of bacteria, mitochondria, and chloroplasts, where they perform a range of structural, transport, and catalytic functions (1). In addition to their biological interest they are increasingly relevant to biotechnology, serving as scaffolds for bacterial surface display (2, 3) and atomically precise pores for nanopore-based DNA sequencing. Although the suitability of natural β-barrel membrane proteins for biotechnology has been improved by protein engineering (3-10), the ability to design membrane proteins de novo would deliver tools customized to meet the demands of each application.De novo design provides a stringent test of our understanding of the determinants of protein folding and stability. Protein design software [e.g., Rosetta (11,12)] has made tremendous strides in addressing the design problem for small water-soluble proteins (13-15), and design of simplified model α-helical membrane proteins including single transmembrane helices and small bundles (16)(17)(18)(19)(20) has also been accomplished. In contrast, a designed β-barrel membrane protein has yet to be reported, perhaps as a consequence of the unique design challenges presented by the folding pathway and architecture of these proteins. Unlike the α-helical membrane proteins, nascent β-barrel membrane proteins must transit the periplasm to the outer membrane, where folding and membrane insertion are thought to occur in concert (21,22). An extensive network of chaperones maintains the solubility of the unfolded barrel and guides membrane insertion. The C-terminal β-strand is known to interact with the BAM chaperone complex (23-25), which assists the folding of all β-barrel membrane proteins. However, despite recent progress (26-30), we do not fully understand how interactions between chaperones and transiting membrane proteins are directed by sequence-encoded information.Further complicating design is the inside-out architecture of β-barrel membrane proteins. In place of a hydrophobic core is either a central water-filled pore or a solid core composed of polar side chains. The lipid bilayer becomes increasingly hydrophobic...