In silico evaluation of the influence of the translocon on partitioning of membrane segments

Background The locations of the TM segments inside the membrane proteins are the consequence of a cascade of several events: the localizing of the nascent chain to the membrane, its insertion through the translocon, and the conformation adopted to reach its stable state inside the lipid bilayer. Even though the hydrophobic h-region of signal peptides and a typical TM segment are both composed of mostly hydrophobic side chains, the translocon has the ability to determine whether a given segment is to be inserted into the membrane. Our goal is to acquire robust biological insights into the influence of the translocon on membrane insertion of helices, obtained from the in silico discrimination between signal peptides and transmembrane segments of bitopic proteins. Therefore, by exploiting this subtle difference, we produce an optimized scale that evaluates the tendency of each amino acid to form sequences destined for membrane insertion by the translocon. Results The learning phase of our approach is conducted on carefully chosen data and easily converges on an optimal solution called the PMIscale (Potential Membrane Insertion scale). Our study leads to two striking results. Firstly, with a very simple sliding-window prediction method, PMIscale enables an efficient discrimination between signal peptides and signal anchors. Secondly, PMIscale is also able to identify TM segments and to localize them within protein sequences. Conclusions Despite its simplicity, the localization method based on PMIscale nearly attains the highest level of TM topography prediction accuracy as the current state-of-the-art prediction methods. These observations confirm the prominent role of the translocon in the localization of TM segments and suggest several biological hypotheses about the physical properties of the translocon

BB0172, a Borrelia burgdorferi Outer Membrane Protein That Binds Integrin Α3Β1

Lyme disease is a multisystemic disorder caused by Borrelia burgdorferi infection. Upon infection, some B. burgdorferi genes are upregulated, including members of the microbial surface components recognizing adhesive matrix molecule (MSCRAMM) protein family, which facilitate B. burgdorferi adherence to extracellular matrix components of the host. Comparative genome analysis has revealed a new family of B. burgdorferi proteins containing the von Willebrand factor A (vWFA) domain. In the present study, we characterized the expression and membrane association of the vWFA domain-containing protein BB0172 by using in vitro transcription/translation systems in the presence of microsomal membranes and with detergent phase separation assays. Our results showed evidence of BB0172 localization in the outer membrane, the orientation of the vWFA domain to the extracellular environment, and its function as a metal ion-dependent integrin-binding protein. This is the first report of a borrelial adhesin with a metal ion-dependent adhesion site (MIDAS) motif that is similar to those observed in eukaryotic integrins and has a similar function

Structural analysis of membrane protein biogenesis and ribosome stalling by cryo-electron microscopy

To study the mechanisms of membrane protein insertion we established a protocol that allows isolation of in vivo assembled ribosome nascent chain complexes (RNCs) from E. coli in high yield and quality. To investigate the interaction of SecY with a translating ribosome, model membrane proteins of different length and topology were over-expressed and the respective RNCs were isolated under mild conditions to allow co-purification of the SecY complex. Analysis of the interaction of RNCs with SecY in vivo suggested that, as expected, a tight engagement of the ribosome and SecY is only established for nascent chains that are translocated co-translationally. We observed that SecY and the RNC do not form a stable complex at the moment of hydrophobic transmembrane segments inserting in the translocon. However, a stable engagement of the RNC with SecY was observed, when inserting a transmembrane segment with a type II topology into SecY followed by a hydrophilic loop of a certain length which allows the isolation of this complex. That suggested a dual binding mode of tight and loose coupling of SecY to the translating ribosome dependent on the nature of the nascent substrate. We present the first three dimensional structure of an in vivo assembled, tightly coupled polytopic RNC-SecYE complex at 7.2 Å solved by cryo-EM and single particle reconstruction. A molecular model based on the cryo-EM structure reveals that SecYE could be trapped in a post-insertion state, with the two substrate helices interacting with the periphery of SecY, while still translocating the hydrophilic loop. The lateral gate of SecY remains in a ‘pre-opened’ conformation during the translocation of the hydrophilic loop. The interaction sites of SecY with the ribosome were found as described. Remarkably, we could also reveal an interaction of helix 59 in the ribosome with nascent membrane protein via positively charged residues in the first cytoplasmic loop of the substrate. It is tempting to speculate that this interaction contributes to the positive inside rule. Though, we provided an unprecedented snapshot of an inserting polytopic membrane protein, the exact path of the nascent chain and the molecular mechanism of the actual insertion could not be solved so far. Expression of the E. coli tryptophanase (TnaA) operon is triggered by ribosome stalling during translation of the upstream TnaC leader peptide. Notably, this stalling is strictly dependent on the presence of tryptophan that acts in a hitherto unknown way. Here, we present a cryo-EM reconstruction of the stalled nascent TnaC leader peptide in the ribosomal exit tunnel. The structure of the TnaC-stalled ribosome was solved to an average resolution of 3.8 Å by cryo-EM and single particle analysis. It reveals the conformation of the silenced peptidyl-transferase center as well as the exact path of the stalled nascent peptide and its contacts in detail. Furthermore, we clearly resolve not a single but two free tryptophan molecules in the ribosomal exit tunnel. The nascent TnaC peptide chain together with distinct rRNA bases in the ribosomal exit tunnel creates two hydrophobic binding pockets for the tryptophan coordination. One tryptophan molecule is coordinated by V20 and I19 of TnaC and interacts with U2586 of the rRNA, the second tryptophan is bound between I19 and I15 in the area of A2058 and A2059 of the rRNA. Interestingly, the latter is also the binding platform for macrolide antibiotics. Engagement of L-Trp in these composite binding pockets leads to subtle conformational changes in residues of the ribosomal tunnel wall that are translated to the PTC eventually resulting in silencing by stabilizing the conformations of the conserved nucleotides A2602 and U2585. These conformations of the two nucleotides in the PTC are incompatible with the correct accommodation of the GGQ motive of release factor 2, thus inhibiting the peptide release

Membrane topology of gp41 and amyloid precursor protein: interfering transmembrane interactions as potential targets for HIV and Alzheimer treatment

The amyloid precursor protein (APP), that plays a critical role in the development of senile plaques in Alzheimer disease (AD), and the gp41 envelope protein of the human immunodeficiency virus (HIV), the causative agent of the acquired immunodeficiency syndrome (AIDS), are single-spanning type-1 transmembrane (TM) glycoproteins with the ability to form homo-oligomers. In this review we describe similarities, both in structural terms and sequence determinants of their TM and juxtamembrane regions. The TM domains are essential not only for anchoring the proteins in membranes but also have functional roles. Both TM segments contain GxxxG motifs that drive TM associations within the lipid bilayer. They also each possess similar sequence motifs, positioned at the membrane interface preceding their TM domains. These domains are known as cholesterol recognition/interaction amino acid consensus (CRAC) motif in gp41 and CRAC-like motif in APP. Moreover, in the cytoplasmic domain of both proteins other alpha-helical membranotropic regions with functional implications have been identified. Recent drug developments targeting both diseases are reviewed and the potential use of TM interaction modulators as therapeutic targets is discussed

Structural analysis of membrane protein biogenesis and ribosome stalling by cryo-electron microscopy

To study the mechanisms of membrane protein insertion we established a protocol that allows isolation of in vivo assembled ribosome nascent chain complexes (RNCs) from E. coli in high yield and quality. To investigate the interaction of SecY with a translating ribosome, model membrane proteins of different length and topology were over-expressed and the respective RNCs were isolated under mild conditions to allow co-purification of the SecY complex. Analysis of the interaction of RNCs with SecY in vivo suggested that, as expected, a tight engagement of the ribosome and SecY is only established for nascent chains that are translocated co-translationally. We observed that SecY and the RNC do not form a stable complex at the moment of hydrophobic transmembrane segments inserting in the translocon. However, a stable engagement of the RNC with SecY was observed, when inserting a transmembrane segment with a type II topology into SecY followed by a hydrophilic loop of a certain length which allows the isolation of this complex. That suggested a dual binding mode of tight and loose coupling of SecY to the translating ribosome dependent on the nature of the nascent substrate. We present the first three dimensional structure of an in vivo assembled, tightly coupled polytopic RNC-SecYE complex at 7.2 Å solved by cryo-EM and single particle reconstruction. A molecular model based on the cryo-EM structure reveals that SecYE could be trapped in a post-insertion state, with the two substrate helices interacting with the periphery of SecY, while still translocating the hydrophilic loop. The lateral gate of SecY remains in a ‘pre-opened’ conformation during the translocation of the hydrophilic loop. The interaction sites of SecY with the ribosome were found as described. Remarkably, we could also reveal an interaction of helix 59 in the ribosome with nascent membrane protein via positively charged residues in the first cytoplasmic loop of the substrate. It is tempting to speculate that this interaction contributes to the positive inside rule. Though, we provided an unprecedented snapshot of an inserting polytopic membrane protein, the exact path of the nascent chain and the molecular mechanism of the actual insertion could not be solved so far. Expression of the E. coli tryptophanase (TnaA) operon is triggered by ribosome stalling during translation of the upstream TnaC leader peptide. Notably, this stalling is strictly dependent on the presence of tryptophan that acts in a hitherto unknown way. Here, we present a cryo-EM reconstruction of the stalled nascent TnaC leader peptide in the ribosomal exit tunnel. The structure of the TnaC-stalled ribosome was solved to an average resolution of 3.8 Å by cryo-EM and single particle analysis. It reveals the conformation of the silenced peptidyl-transferase center as well as the exact path of the stalled nascent peptide and its contacts in detail. Furthermore, we clearly resolve not a single but two free tryptophan molecules in the ribosomal exit tunnel. The nascent TnaC peptide chain together with distinct rRNA bases in the ribosomal exit tunnel creates two hydrophobic binding pockets for the tryptophan coordination. One tryptophan molecule is coordinated by V20 and I19 of TnaC and interacts with U2586 of the rRNA, the second tryptophan is bound between I19 and I15 in the area of A2058 and A2059 of the rRNA. Interestingly, the latter is also the binding platform for macrolide antibiotics. Engagement of L-Trp in these composite binding pockets leads to subtle conformational changes in residues of the ribosomal tunnel wall that are translated to the PTC eventually resulting in silencing by stabilizing the conformations of the conserved nucleotides A2602 and U2585. These conformations of the two nucleotides in the PTC are incompatible with the correct accommodation of the GGQ motive of release factor 2, thus inhibiting the peptide release

Mechanisms of ER Protein Import

Protein import into the endoplasmic reticulum (ER) is the first step in the biogenesis of approximately 10,000 different soluble and membrane proteins of human cells, which amounts to about 30% of the proteome. Most of these proteins fulfill their functions either in the membrane or lumen of the ER plus the nuclear envelope, in one of the organelles of the pathways for endo- and exocytosis (ERGIC, Golgi apparatus, endosome, lysosome, and trafficking vesicles), or at the cell surface as plasma membrane or secreted proteins. An increasing number of membrane proteins destined to lipid droplets, peroxisomes or mitochondria are first targeted to and inserted into the ER membrane prior to their integration into budding lipid droplets or peroxisomes or prior to their delivery to mitochondria via the ER-SURF pathway. ER protein import involves two stages, ER targeting, which guarantees membrane specificity, and the insertion of nascent membrane proteins into or translocation of soluble precursor polypeptides across the ER membrane. In most cases, both processes depend on amino-terminal signal peptides or transmembrane helices, which serve as signal peptide equivalents. However, the targeting reaction can also involve the ER targeting of specific mRNAs or ribosome–nascent chain complexes. Both processes may occur co- or post-translationally and are facilitated by various sophisticated machineries, which reside in the cytosol and the ER membrane, respectively. Except for resident ER and mitochondrial membrane proteins, the mature proteins are delivered to their functional locations by vesicular transport

Towards Understanding How Membrane Proteins Approach And Fold Into Membranes

Membrane proteins must fold into phospholipid bilayers to function. Some membrane proteins are cotranslationally inserted into the membrane, while others rely on chaperone networks to solubilize and traffic unfolded membrane proteins to the membrane. In my thesis work I have studied both facets of membrane protein folding: chaperone interactions and insertion into the membrane. The first part of my thesis investigates the intrinsic conformational properties and unfolded outer membrane protein (uOMP) binding of the main chaperone in OMP biogenesis pathway in E. coli: SurA. We found that SurA is monomeric and exists in three major conformations in solution. Next, we mapped the uOMP binding site on SurA, finding that the least intrinsically populated conformation is the chaperone-active state. Using a plethora of experimental data as restraints, we constructed a model of the SurA-uOMP complex in which the uOMP is greatly expanded by SurA. In the second part of my thesis I examined the effect of the local environment imposed by both the protein and bilayer on individual side chain transfer free energies. I found that the transfer free energies for most side chains are only slightly altered by changes in neighboring side chains and packing. By relating the local composition of the membrane to nonpolar side chain transfer free energies, I discovered a linear correlation between the nonpolar solvation parameter and local concentration of water in the bilayer. Together these studies highlight the structural and thermodynamic parameters that drive efficient membrane protein biogenesis and folding

Membrane bending is critical for assessing the thermodynamic stability of proteins in the membrane

The ability of biological membranes to bend is critical to understanding the interaction between proteins and the lipid bilayer. Experimental and computational studies have shown that the membrane can bend to expose charged and polar residues to the lipid headgroups and water, greatly reducing the cost of protein insertion. However, current computational approaches are poorly equipped to accurately model such deformation; atomistic simulations often do not reach the time-scale necessary to observe large-scale rearrangement, and continuum approaches assume a flat, rigid bilayer. In this thesis we present an efficient computational model of a deformable membrane for probing these interactions with elasticity theory and continuum electrostatics. To validate the model, we first investigate the insertion of three membrane proteins and three aqueous proteins. The model finds the membrane proteins and aqueous proteins stable and unstable in the membrane, respectively. We also investigate the sensitivity of these predictions to changes in several key parameters. The model is then applied to interactions between the membrane and the voltage sensor segments of voltage-gated potassium channels. Despite their high numbers of basic residues, experiments have shown that voltage sensors can be stably accommodated in the membrane. For simple continuum electrostatics approaches that assume a flat membrane, the penalty of inserting these charged residues would seem to prohibit voltage sensor insertion. However, in our method the membrane deforms to enable interaction between solvent and the charged residues. Our calculations predict that the highly charged S4 helices of several potassium channels are in fact stable in the membrane, in accord with experimental observations. Experimental and computational evidence has shown that the cost for inserting multiple charged amino acids into the membrane is not additive; it is not as costly to insert a second charge once a first has already been inserted. Our model reflects this phenomenon and provides a simple mechanical explanation linked to membrane deformation. We additionally consider the energetics of passive ion penetration into the membrane from bulk solvent. We use coarse-grained molecular dynamics to guide our input parameters and show that ion permeation energy profiles agree with atomistic simulations when membrane bending is included