Alessandro Senes

The Cα—H⋅⋅⋅O hydrogen bond: A determinant of stability and specificity in transmembrane helix interactions

The Cα—H⋅⋅⋅O hydrogen bond has been given little attention as a determinant of transmembrane helix association. Stimulated by recent calculations suggesting that such bonds can be much stronger than has been supposed, we have analyzed 11 known membrane protein structures and found that apparent carbon α hydrogen bonds cluster frequently at glycine-, serine-, and threonine-rich packing interfaces between transmembrane helices. Parallel right-handed helix–helix interactions appear to favor Cα—H⋅⋅⋅O bond formation. In particular, Cα—H⋅⋅⋅O interactions are frequent between helices having the structural motif of the glycophorin A dimer and the GxxxG pair. We suggest that Cα—H⋅⋅⋅O hydrogen bonds are important determinants of stability and, depending on packing, specificity in membrane protein folding.

Membrane protein folding: beyond the two stage model

The folding of α‐helical membrane proteins has previously been described using the two stage model, in which the membrane insertion of independently stable α‐helices is followed by their mutual interactions within the membrane to give higher order folding and oligomerization. Given recent advances in our understanding of membrane protein structure it has become apparent that in some cases the model may not fully represent the folding process. Here we present a three stage model which gives considerations to ligand binding, folding of extramembranous loops, insertion of peripheral domains and the formation of quaternary structure.

De novo design and molecular assembly of a transmembrane diporphyrin-binding protein complex

The de novo design of membrane proteins remains difficult despite recent advances in understanding the factors that drive membrane protein folding and association. We have designed a membrane protein PRIME (PoRphyrins In MEmbrane) that positions two non-natural iron diphenylporphyrins (Fe(III)DPPs) sufficiently close to provide a multicentered pathway for transmembrane electron transfer. Computational methods previously used for the design of multiporphyrin water-soluble helical proteins were extended to this membrane target. Four helices were arranged in a D(2)-symmetrical bundle to bind two Fe(II/III) diphenylporphyrins in a bis-His geometry further stabilized by second-shell hydrogen bonds. UV-vis absorbance, CD spectroscopy, analytical ultracentrifugation, redox potentiometry, and EPR demonstrate that PRIME binds the cofactor with high affinity and specificity in the expected geometry.

Inside-out Ca2+ signalling prompted by STIM1 conformational switch

Store-operated Ca2+ entry mediated by STIM1 and ORAI1 constitutes one of the major Ca2+ entry routes in mammalian cells. The molecular choreography of STIM1–ORAI1 coupling is initiated by endoplasmic reticulum (ER) Ca2+ store depletion with subsequent oligomerization of the STIM1 ER-luminal domain, followed by its redistribution towards the plasma membrane to gate ORAI1 channels. The mechanistic underpinnings of this inside-out Ca2+ signalling were largely undefined. By taking advantage of a unique gain-of-function mutation within the STIM1 transmembrane domain (STIM1-TM), here we show that local rearrangement, rather than alteration in the oligomeric state of STIM1-TM, prompts conformational changes in the cytosolic juxtamembrane coiled-coil region. Importantly, we further identify critical residues within the cytoplasmic domain of STIM1 (STIM1-CT) that entail autoinhibition. On the basis of these findings, we propose a model in which STIM1-TM reorganization switches STIM1-CT into an extended conformation, thereby projecting the ORAI-activating domain to gate ORAI1 channels.

Consensus motif for integrin transmembrane helix association

Interactions between transmembrane (TM) helices play an important role in the regulation of diverse biological functions. For example, the TM helices of integrins are believed to interact heteromerically in the resting state; disruption of this interaction results in integrin activation and cellular adhesion. However, it has been difficult to demonstrate the specificity and affinity of the interaction between integrin TM helices and to relate them to the activation process. To examine integrin TM helix associations, we developed a bacterial reporter system and used it to define the sequence motif required for helix–helix interactions in the β1 and β3 integrin subfamilies. The helices interact in a novel three-dimensional motif, the “reciprocating large-small motif” that is also observed in the crystal structures of unrelated proteins. Modest but specific stabilization of helix associations is realized via packing of complementary small and large groups on neighboring helices. Mutations destabilizing this motif activate native, full-length integrins. Thus, this highly conserved dissociable motif plays a vital and widespread role as an on-off switch that can integrate with other control elements during integrin activation.

Amide vibrations are delocalized across the hydrophobic interface of a transmembrane helix dimer

The tertiary interactions between amide-I vibrators on the separate helices of transmembrane helix dimers were probed by ultrafast 2D vibrational photon echo spectroscopy. The 2D IR approach proves to be a useful structural method for the study of membrane-bound structures. The 27-residue human erythrocyte protein Glycophorin A transmembrane peptide sequence: KKITLIIFG79VMAGVIGTILLISWG94IKK was labeled at G79 and G94 with 13C16O or 13C18O. The isotopomers and their 50:50 mixtures formed helical dimers in SDS micelles whose 2D IR spectra showed components from homodimers when both helices had either 13C16O or 13C18O substitution and a heterodimer when one had 13C16O substitution and the other had 13C18O substitution. The cross-peaks in the pure heterodimer 2D IR difference spectrum and the splitting of the homodimer peaks in the linear IR spectrum show that the amide-I mode is delocalized across a pair of helices. The excitation exchange coupling in the range 4.3–6.3 cm−1 arises from through-space interactions between amide units on different helices. The angle between the two Gly79 amide-I transition dipoles, estimated at 103° from linear IR spectroscopy and 110° from 2D IR spectroscopy, combined with the coupling led to a structural picture of the hydrophobic interface that is remarkably consistent with results from NMR on helix dimers. The helix crossing angle in SDS is estimated at 45°. Two-dimensional IR spectroscopy also sets limits on the range of geometrical parameters for the helix dimers from an analysis of the coupling constant distribution.

Computational analysis of membrane proteins: genomic occurrence, structure prediction and helix interactions

We review recent computational advances in the study of membrane proteins, focusing on those that have at least one transmembrane helix. Transmembrane protein regions are, in many respects, easier to investigate computationally than experimentally, due to the uniformity of their structure and interactions (e.g. consisting predominately of nearly parallel helices packed together) on one hand and presenting the challenges of solubility on the other. We present the progress made on identifying and classifying membrane proteins into families, predicting their structure from amino-acid sequence patterns (using many different methods), and analyzing their interactions and packing The total result of this work allows us for the first time to begin to think about the membrane protein interactome, the set of all interactions between distinct transmembrane helices in the lipid bilayer.

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

Significance The transmembrane helices of single-span membrane proteins are commonly engaged in oligomeric interactions that are essential for structure and function. These interactions often occur in the form of recurrent structural motifs. Here we present an analysis of one of the most important motifs (GASright), showing that its geometry is optimized to form carbon hydrogen bonds at the helix−helix interface. The analysis reveals the structural basis for its characteristic GxxxG sequence signature. We built upon the analysis, creating a method that predicts known GASright structures at near-atomic precision. The work has implications for understanding membrane protein association, and for the prediction of unknown interacting GASright dimers among the thousands of single-span proteins in the proteomes of humans and higher organisms. 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, GASright, whose best-known example is glycophorin A. The finding suggests a causal link between the high frequency of occurrence of GASright 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 non-Gly 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 GASright 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 GASright motifs at near-atomic level.

Using α-Helical Coiled-Coils to Design Nanostructured Metalloporphyrin Arrays

We have developed a computational design strategy based on the alpha-helical coiled-coil to generate modular peptide motifs capable of assembling into metalloporphyrin arrays of varying lengths. The current study highlights the extension of a two-metalloporphyrin array to a four-metalloporphyrin array through the incorporation of a coiled-coil repeat unit. Molecular dynamics simulations demonstrate that the initial design evolves rapidly to a stable structure with a small rmsd compared to the original model. Biophysical characterization reveals elongated proteins of the desired length, correct cofactor stoichiometry, and cofactor specificity. The successful extension of the two-porphyrin array demonstrates how this methodology serves as a foundation to create linear assemblies of organized electrically and optically responsive cofactors.

The Oligomeric States of the Purified Sigma-1 Receptor Are Stabilized by Ligands

Background: Sigma-1 receptor (S1R) is an integral membrane ligand-binding receptor. Results: Gel filtration chromatography revealed oligomeric states that are stabilized by ligand binding and destabilized by mutations in the GXXXG integral membrane dimerization domain. Conclusion: Purified S1R binds small molecule ligands as an oligomer but not as a monomer. Significance: The results provide new insight into the function of S1R with ligands and proteins partners. Sigma-1 receptor (S1R) is a mammalian member of the ERG2 and sigma-1 receptor-like protein family (pfam04622). It has been implicated in drug addiction and many human neurological disorders, including Alzheimer and Parkinson diseases and amyotrophic lateral sclerosis. A broad range of synthetic small molecules, including cocaine, (+)-pentazocine, haloperidol, and small endogenous molecules such as N,N-dimethyltryptamine, sphingosine, and steroids, have been identified as regulators of S1R. However, the mechanism of activation of S1R remains obscure. Here, we provide evidence in vitro that S1R has ligand binding activity only in an oligomeric state. The oligomeric state is prone to decay into an apparent monomeric form when exposed to elevated temperature, with loss of ligand binding activity. This decay is suppressed in the presence of the known S1R ligands such as haloperidol, BD-1047, and sphingosine. S1R has a GXXXG motif in its second transmembrane region, and these motifs are often involved in oligomerization of membrane proteins. Disrupting mutations within the GXXXG motif shifted the fraction of the higher oligomeric states toward smaller states and resulted in a significant decrease in specific (+)-[3H]pentazocine binding. Results presented here support the proposal that S1R function may be regulated by its oligomeric state. Possible mechanisms of molecular regulation of interacting protein partners by S1R in the presence of small molecule ligands are discussed.