Abstract: Colocalization single-molecule methods can provide a wealth of information concerning the ordering and dynamics of biomolecule assembly. These have been used extensively to study the pathways of spliceosome assembly in vitro. Key to these experiments is the measurement of binding times-either the dwell times of a multi-molecular interaction or times in between binding events. By analyzing hundreds of these times, many new insights into the kinetic pathways governing spliceosome assembly have been obtained. Collections of binding times are often plotted as histograms and can be fit to kinetic models using a variety of methods. Here, we describe the use of maximum likelihood methods to fit dwell time distributions without binning. In addition, we discuss several aspects of analyzing these distributions with histograms and pitfalls that can be encountered if improperly binned histograms are used. We have automated several aspects of maximum likelihood fitting of dwell time distributions in the AGATHA software package.

Abstract: Degradation of the plant hormone cytokinin is controlled by cytokinin oxidase/dehydrogenase (CKX) enzymes. The molecular and cellular behavior of these proteins is still largely unknown. In this study, we show that CKX1 is a type-II single-pass membrane protein that predominantly localizes to the endoplasmic reticulum (ER). This indicates that this CKX isoform is a bona fide ER protein directly controlling the cytokinin which triggers the signaling from the ER. By using various approaches, we demonstrate that CKX1 forms homodimers and homooligomers in vivo. The N-terminal part of CKX1 was necessary and sufficient for the protein oligomerization as well as for targeting and retention in the ER. Moreover, we show that protein-protein interaction is largely facilitated by transmembrane helices and depends on a functional GxxxG-like interaction motif. Importantly, mutations rendering CKX1 monomeric interfere with its steady-state localization in the ER and cause a loss of the CKX1 biological activity by increasing its ER-associated degradation. Therefore, our study provides evidence that oligomerization is a crucial parameter regulating CKX1 biological activity and the cytokinin concentration in the ER. The work also lends a strong support for the cytokinin signaling from the ER and for the functional relevance of the cytokinin pool in this compartment.

Abstract: In Escherichia coli, FtsLB plays a central role in the initiation of cell division, possibly transducing a signal that will eventually lead to the activation of peptidoglycan remodeling at the forming septum. The molecular mechanisms by which FtsLB operates in the divisome, however, are not understood. Here we present a structural analysis of the FtsLB complex — performed with biophysical, computational and in vivo methods — that establishes the organization of the transmembrane region and proximal coiled coil of the complex. FRET analysis in vitro is consistent with formation of a tetramer composed of two FtsL and two FtsB subunits. We predicted subunit contacts through co-evolutionary analysis and used them to compute a structural model of the complex. The transmembrane region of FtsLB is stabilized by hydrophobic packing and by a complex network of hydrogen bonds. The coiled coil domain likely terminates near the critical Constriction Control Domain, which might correspond to a structural transition. Presence of strongly polar amino acids within the core of the tetrameric coiled coil suggests that the coil may split into two independent FtsQ-binding domains. The helix of FtsB is interrupted between the transmembrane and coiled coil regions by a flexible Gly-rich linker. Conversely, the data suggest that FtsL forms an uninterrupted helix across the two regions, and that integrity of this helix is indispensable for the function of the complex. The FtsL helix is thus a candidate for acting as a potential mechanical connection to communicate conformational changes between periplasmic, membrane, and cytoplasmic regions.

Abstract: The GxxxG motif is frequently found at the dimerization interface of a transmembrane structural motif called GAS_right, which is characterized by a short interhelical distance and a right-handed crossing angle between the helices. In GAS_right dimers, such as glycophorin A (GpA), BNIP3, and members of the ErbB family, the backbones of the helices are in contact, and they invariably display networks of 4 to 8 weak hydrogen bonds between Cα-H carbon donors and carbonyl acceptors on opposing helices (Cα-H···O=C hydrogen bonds). These networks of weak hydrogen bonds at the helix-helix interface are presumably stabilizing, but their energetic contribution to dimerization has yet to be determined experimentally. Here, we present a computational and experimental structure-based analysis of GAS_right dimers of different predicted stabilities, which show that a combination of van der Waals packing and Cα-H hydrogen bonding predicts the experimental trend of dimerization propensities. This finding provides experimental support for the hypothesis that the networks of Cα-H hydrogen bonds are major contributors to the free energy of association of GxxxG-mediated dimers. The structural comparison between groups of GAS_right dimers of different stabilities reveals distinct sequence as well as conformational preferences. Stability correlates with shorter interhelical distances, narrower crossing angles, better packing, and the formation of larger networks of Cα-H hydrogen bonds. The identification of these structural rules provides insight on how nature could modulate stability in GAS_right and finely tune dimerization to support biological function.

Abstract: TOXCAT is a widely used genetic assay to study interactions of transmembrane helices within the inner membrane of the bacterium Escherichia coli. TOXCAT is based on a fusion construct that links a transmembrane domain of interest with a cytoplasmic DNA-binding domain from the Vibrio cholerae ToxR protein. Interaction driven by the transmembrane domain results in dimerization of the ToxR domain, which, in turn, activates the expression of the reporter gene chloramphenicol acetyl transferase (CAT). Quantification of CAT is used as a measure of the ability of the transmembrane domain to self-associate. Because the quantification of CAT is relatively laborious, we developed a high-throughput variant of the assay, TOXGREEN, based on the expression of super-folded GFP and detection of fluorescence directly in unprocessed cell cultures. Careful side-by-side comparison of TOXCAT and TOXGREEN demonstrates that the methods have comparable response, dynamic range, sensitivity and intrinsic variability both in LB and minimal media. The greatly enhanced workflow makes TOXGREEN much more scalable and ideal for screening, since hundreds of constructs can be rapidly assessed in 96 well plates. Even for small scale investigations, TOXGREEN significantly reduces time, labor and cost associated with the procedure. We demonstrate applicability with a large screening for self-association among the transmembrane domains of bitopic proteins of the divisome (FtsL, FtsB, FtsQ, FtsI, FtsN, ZipA and EzrA) belonging to 11 bacterial species. The analysis confirms a previously reported tendency for FtsB to self-associate, and suggests that the transmembrane domains of ZipA, EzrA and FtsN may also possibly oligomerize.

Abstract: The computational design of α-helical membrane proteins is still in its infancy but has already made great progress. De novo design allows stable, specific and active minimal oligomeric systems to be obtained. Computational reengineering can improve the stability and function of naturally occurring membrane proteins. Currently, the major hurdle for the field is the experimental characterization of the designs. The emergence of new structural methods for membrane proteins will accelerate progress.

Abstract: Pro-apoptotic Bax induces mitochondrial outer membrane permeabilization (MOMP) by forming oligomers through a largely undefined process. Using site-specific disulfide crosslinking, compartment-specific chemical labeling, and mutational analysis, we found that activated integral membrane Bax proteins form a BH3-in-groove dimer interface on the MOM surface similar to that observed in crystals. However, after the α5 helix was released into the MOM, the remaining interface with α2, α3, and α4 helices was rearranged. Another dimer interface was formed inside the MOM by two intersected or parallel α9 helices. Combinations of these interfaces generated oligomers in the MOM. Oligomerization was initiated by BH3-in-groove dimerization, without which neither the other dimerizations nor MOMP occurred. In contrast, α9 dimerization occurred downstream and was required for release of large but not small proteins from mitochondria. Moreover, the release of large proteins was facilitated by α9 insertion into the MOM and localization to the pore rim. Therefore, the BH3-in-groove dimerization on the MOM nucleates the assembly of an oligomeric Bax pore that is enlarged by α9 dimerization at the rim.

Abstract: FRET has been widely used as a spectroscopic tool in vitro to study the interactions between transmembrane helices in detergent and lipid environments. This technique has been instrumental to many studies that have greatly contributed to quantitative understanding of the physical principles that govern helix-helix interaction in the membrane. These studies have also improved our understanding of the biological role of oligomerization in membrane proteins. In this review, we focus on the combinations of fluorophores used, the membrane mimetic environments and measurement techniques that have been applied to study model systems as well as biological oligomeric complexes in vitro. We highlight the different formalisms used to calculate FRET efficiency, and the challenges associated with accurate quantification. The goal is to provide the reader with a comparative summary of the relevant literature for planning and designing FRET experiments aimed at measuring transmembrane helix-helix association.

Abstract: 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 toward the plasma membrane to gate ORAI1 channels. The mechanistic underpinnings of this inside-out Ca²⁺ signaling 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. Our findings provide compelling evidence to support a conformational switch model in which STIM1-TM reorganization switches STIM1-CT into an extended conformation, thereby unleashing the ORAI-activating domain to gate ORAI1 channels.

Abstract: Interactions between α-helices within the hydrophobic environment of lipid bilayers are integral to the folding and function of transmembrane proteins; however, the major forces that mediate these interactions remain debated, and our ability to predict these interactions is still largely untested. We recently demonstrated that the frequent transmembrane association motif GAS_right - the GxxxG-containing fold of the glycophorin A dimer - is optimal for the formation of extended networks of Cα-H hydrogen bonds, supporting the hypothesis that these bonds are major contributors to association. We also found that optimization of Cα-H hydrogen bonding and inter-helical packing is sufficient to computationally predict the structure of known GAS_right dimers at near atomic level. Here, we demonstrate that this computational method can be used to characterize the structure of a protein not previously known to dimerize, by predicting and validating the transmembrane dimer of ADCK3, a mitochondrial kinase. ADCK3 is involved in the biosynthesis of the redox active lipid, ubiquinone, and human ADCK3 mutations cause a cerebellar ataxia associated with ubiquinone deficiency, but the biochemical functions of ADCK3 remain largely undefined. Our experimental analyses show that the transmembrane helix of ADCK3 dimerizes, with an interface based on an extended Gly-zipper motif, as predicted by our models. The data provide strong evidence for the hypothesis that optimization of Cα-H hydrogen bonding is an important factor in the association of transmembrane helices. This work also provides a structural foundation for investigating the role of transmembrane association in regulating the biological activity of ADCK3.

Abstract: Side chain optimization is an integral component of many protein modeling applications. In these applications, the conformational freedom of the side chains is often explored using libraries of discrete, frequently occurring conformations. Because side chain optimization can pose a computationally intensive combinatorial problem, the nature of these conformer libraries is important for ensuring efficiency and accuracy in side chain prediction. We have previously developed an innovative method to create a conformer library with enhanced performance. The Energy-Based Library (EBL) was obtained by analyzing the energetic interactions between conformers and a large number of natural protein environments from crystal structures. This process guided the selection of conformers with the highest propensity to fit into spaces that should accommodate a side chain. Because the method requires a large crystallographic data-set, the EBL was created in a backbone-independent fashion. However, it is well established that side chain conformation is strongly dependent on the local backbone geometry, and that backbone-dependent libraries are more efficient in side chain optimization. Here we present a backbone-dependent version of the EBL, the bEBL, whose conformers are independently sorted for each populated region of Ramachandran space. The resulting library closely mirrors the local backbone-dependent distribution of side chain conformation. Compared to the EBL, we demonstrate that the bEBL uses fewer conformers to produce similar side chain prediction outcomes, thus further improving performance with respect to the already efficient backbone-independent version of the library.

Abstract: Sigma 1 receptor (S1R) is a mammalian member of the ERG2 and sigma1 receptor like protein family (pfam04622). It has been implicated in drug addiction and many human neurological disorders including Alzheimers and Parkinsons 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 towards 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.

Abstract: 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 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 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.

Abstract: FtsB and FtsL are two essential integral membrane proteins of the bacterial division complex or "divisome", both characterized by a single transmembrane helix and a juxtamembrane coiled coil domain. The two domains are important for the association of FtsB and FtsL, a key event for their recruitment to the divisome, which in turn allows the recruitment of the late divisomal components to the Z-ring and subsequent completion of the division process. Here we present a biophysical analysis performed in vitro that shows that the transmembrane domains of FtsB and FtsL associate strongly in isolation. Using Förster resonance energy transfer, we have measured the oligomerization of fluorophore-labeled transmembrane domains of FtsB and FtsL in both detergent and lipid. The data indicate that the transmembrane helices are likely a major contributor to the stability of the FtsB−FtsL complex. Our analyses show that FtsB and FtsL form a 1:1 higher-order oligomeric complex, possibly a tetramer. This finding suggests that the FtsB−FtsL complex is capable of multivalent binding to FtsQ and other divisome components, a hypothesis that is consistent with the possibility that the FtsB−FtsL complex has a structural role in the stabilization of the Z-ring.

Abstract: Present day understanding of the thermodynamic properties of integral membrane proteins (IMPs) lags behind that of water-soluble proteins due to difficulties in mimicking the physiological environment of the IMPs in order to obtain a reversible folded system. Despite such challenges faced in studying these systems, significant progress has been made in the study of the oligomerization of single span transmembrane helices. One of the primary methods available to characterize these systems is based on Förster Resonance Energy Transfer (FRET). FRET is a widely used spectroscopic tool that provides proximity data that can be fitted to obtain the energetics of a system. Here we discuss various technical aspects related to the application of FRET to study transmembrane peptide oligomerization in liposomes. The analysis is based on FRET efficiency relative to the concentration of the peptides in the bilayer (peptide:lipid ratio). Some important parameters that will be discussed include labeling efficiency, sample homogeneity and equilibration. Furthermore, data analysis has to be performed keeping in mind random colocalization of donors and acceptors in liposome vesicles.

Abstract: We report the first structural analysis of an integral membrane protein of the bacterial divisome. FtsB is a single-pass membrane protein with a periplasmic coiled coil. Its heterologous association with its partner FtsL represents an essential event for the recruitment of the late components to the division site. Using a combination of mutagenesis, computational modeling and X-ray crystallography, we determined that FtsB self-associates and we investigated its structural organization. We found that the transmembrane domain of FtsB homo-oligomerizes through an evolutionarily conserved interaction interface where a polar residue (Gln 16) plays a critical role through the formation of an inter-helical hydrogen bond. The crystal structure of the periplasmic domain, solved as a fusion with Gp7, shows that 30 juxta-membrane amino acids of FtsB form a canonical coiled coil. The presence of conserved Gly residue in the linker region suggests that flexibility between the transmembrane and coiled coil domains is functionally important. We hypothesize that the transmembrane helices of FtsB form a stable dimeric core for its association with FtsL into a higher-order oligomer, and that FtsL is required to stabilize the periplasmic domain of FtsB, leading to the formation of a complex that is competent for binding to FtsQ, and to their consequent recruitment to the divisome. The study provides an experimentally validated structural model and identifies point mutations that disrupt association, thereby establishing important groundwork for the functional characterization of FtsB in vivo.

Abstract: Side chain optimization is a fundamental component of protein modeling applications such as docking, structural prediction, and design. In these applications side chain flexibility is often provided by rotamer or conformer libraries, which are collections of representative side chain conformations. Here we demonstrate that the sampling provided by the library can be substantially improved by adding an energetic criterion to its creation. The result of the new procedure is the Energy-Based library, a conformer library selected according to the propensity of its elements to fit energetically into natural protein environments. The new library performs outstandingly well in side chain optimization, producing structures with significantly lower energies and resulting in improved side chain conformation prediction. In addition, because the library was created as an ordered list, its size can be adjusted to any desired level. This feature provides unprecedented versatility in tuning sampling. It allows to precisely balance the number of conformers required by each amino acid type, equalizing their chances to fit into structural environments. It also allows to scale the amount of sampling to the specific requirement of any given side optimization problem. A rotameric version of the library was also produced with the same method to support applications that require a dihedral-only description of side chain conformation. The libraries are available at http://seneslab.org/EBL.

Abstract: We present the Molecular Software Library (MSL), a C++ library for molecular modeling. MSL is a set of tools that supports a large variety of algorithms for the design, modeling, and analysis of macromolecules. Among the main features supported by the library are methods for applying geometric transformations and alignments, the implementation of a rich set of energy functions, side chain optimization, backbone manipulation, calculation of solvent accessible surface area, and other tools. MSL has a number of unique features, such as the ability of storing alternative atomic coordinates (for modeling) and multiple amino acid identities at the same backbone position (for design). It has a straightforward mechanism for extending its energy functions and can work with any type of molecules. Although the code base is large, MSL was created with ease of developing in mind. It allows the rapid implementation of simple tasks while fully supporting the creation of complex applications. Some of the potentialities of the software are demonstrated here with examples that show how to program complex and essential modeling tasks with few lines of code. MSL is an ongoing and evolving project, with new features and improvements being introduced regularly, but it is mature and suitable for production and has been used in numerous protein modeling and design projects. MSL is open-source software, freely downloadable at http://msl-libraries.org. We propose it as a common platform for the development of new molecular algorithms and to promote the distribution, sharing, and reutilization of computational methods.

Abstract: In the Rhodobacter (Rba.) species of photosynthetic purple bacteria, a single transmembrane α-helix, PufX, is found within the core complex, an essential photosynthetic macromolecular assembly that performs the absorption and the initial processing of light energy. Despite its structural simplicity, many unresolved questions surround PufX, the most important of which is its location within the photosynthetic core complex. One proposed placement of PufX is at the center of a core complex dimer, where two PufX helices associate in the membrane and form a homodimer. Inability for PufX of certain Rba. species to form a homodimer is thought to lead to monomeric core complexes. In the present study, we employ a combination of computational and experimental techniques to test the hypothesized homodimerization of PufX. We carry out a systematic investigation to measure the dimerization affinity of PufX from four Rba. species, Rba. blasticus , Rba. capsulatus , Rba. sphaeroides , and Rba. veldkampii , using a molecular dynamics-based free-energy method, as well as experimental TOXCAT assays. We found that the four PufX helices have substantially different dimerization affinities. Both computational and experimental techniques demonstrate that species with dimeric core complexes have PufX that can potentially form a homodimer, whereas the one species with monomeric core complexes has a PufX with little to no dimerization propensity. Our analysis of the helix-helix interface revealed a number of positions that may be important for PufX dimerization and the formation of a hydrogen-bond network between these GxxxG-containing helices. Our results suggest that the different oligomerization states of core complexes in various Rba. species can be attributed, among other factors, to the different propensity of its PufX helix to homodimerize.

Abstract: This article reviews the recent successes of computational protein design techniques applied to integral membrane proteins. This emerging area is still handicapped by significant difficulties in the experimental characterization of the stability and structure of the designed proteins. Nevertheless, by focusing on oligomeric complexes of single-span transmembrane (TM) peptides with detectable activity, the computational design of membrane proteins has already produced very exciting results. The 'take-home message' is that optimization of van der Waals packing and hydrogen bonding (both 'canonical' and weak Cα-H⋯O bonds) can produce functional structures of remarkable stability and specificity in the membrane.

Abstract: Side chain optimization is the process of packing the sidechains of a protein onto a fixed backbone structure, such that the energy of the resultant structure is minimized. The continuous space of sidechain conformations is typically handled by discretizing (sampling) into a finite set of representative conformations called a "conformer library". The key contribution of this work is to use machine learning methods to distribute (conformational) sampling among different positions in a protein. The idea is that different positions in a protein backbone have different sampling requirements, for example, solvent exposed positions require less sampling than positions in the core of a protein. We propose a 3-ary categorization of every position in a target protein based on its sampling requirements and evaluate it by comparing against an unbiased distribution of conformers. Our results demonstrate that this strategy helps to distribute the sampling more efficiently for sidechain optimization.