Determining the helical tilt angle and dynamic properties of the transmembrane domains of pinholin S2168 using mechanical alignment EPR spectroscopy

The lytic cycle of bacteriophage φ21 for the infected E. coli is initiated by pinholin S²¹, which determines the timing of host cell lysis through the function of pinholin (S²¹68) and antipinholin (S²¹71). The activity of pinholin or antipinholin directly depends on the function of two transmembrane domains (TMDs) within the membrane. For active pinholin, TMD1 externalizes and lies on the surface while TMD2 remains incorporated inside the membrane forming the lining of the small pinhole. In this study, spin labeled pinholin TMDs were incorporated separately into mechanically aligned POPC (1-palmitoyl-2-oleoyl-glycero-3-phosphocholine) lipid bilayers and investigated with electron paramagnetic resonance (EPR) spectroscopy to determine the topology of both TMD1 and TMD2 with respect to the lipid bilayer; the TOAC (2,2,6,6-tetramethyl-N-oxyl-4-amino-4-carboxylic acid) spin label was used here because it attaches to the backbone of a peptide and is very rigid. TMD2 was found to be nearly colinear with the bilayer normal (n) with a helical tilt angle of 16 ± 4° while TMD1 lies on or near the surface with a helical tilt angle of 84 ± 4°. The order parameters (~0.6 for both TMDs) obtained from our alignment study were reasonable, which indicates the samples incorporated inside the membrane were well aligned with respect to the magnetic field (B₀). The data obtained from this study supports previous findings on pinholin: TMD1 partially externalizes from the lipid bilayer and interacts with the membrane surface, whereas TMD2 remains buried in the lipid bilayer in the active conformation of pinholin S²¹68. In this study, the helical tilt angle of TMD1 was measured for the first time. For TMD2 our experimental data corroborates the findings of the previously reported helical tilt angle by the Ulrich group.     

Pinholin S21 mutations induce structural topology and conformational changes

The bacteriophage infection cycle is terminated at a predefined time to release the progeny virions via a robust lytic system composed of holin, endolysin, and spanin proteins. Holin is the timekeeper of this process. Pinholin S²¹ is a prototype holin of phage Φ21, which determines the timing of host cell lysis through the coordinated efforts of pinholin and antipinholin. However, mutations in pinholin and antipinholin play a significant role in modulating the timing of lysis depending on adverse or favorable growth conditions. Earlier studies have shown that single point mutations of pinholin S²¹ alter the cell lysis timing, a proxy for pinholin function as lysis is also dependent on other lytic proteins. In this study, continuous wave electron paramagnetic resonance (CW-EPR) power saturation and double electron-electron resonance (DEER) spectroscopic techniques were used to directly probe the effects of mutations on the structure and conformational changes of pinholin S²¹ that correlate with pinholin function. DEER and CW-EPR power saturation data clearly demonstrate that increased hydrophilicity induced by residue mutations accelerate the externalization of antipinholin transmembrane domain 1 (TMD1), while increased hydrophobicity prevents the externalization of TMD1. This altered hydrophobicity is potentially accelerating or delaying the activation of pinholin S²¹. It was also found that mutations can influence intra- or intermolecular interactions in this system, which contribute to the activation of pinholin and modulate the cell lysis timing. This could be a novel approach to analyze the mutational effects on other holin systems, as well as any other membrane protein in which mutation directly leads to structural and conformational changes.     

Probing the local secondary structure of bacteriophage S21 pinholin membrane protein using electron spin echo envelope modulation spectroscopy

There have recently been advances in methods for detecting local secondary structures of membrane protein using electron paramagnetic resonance (EPR). A three pulsed electron spin echo envelope modulation (ESEEM) approach was used to determine the local helical secondary structure of the small hole forming membrane protein, S²¹ pinholin. This ESEEM approach uses a combination of site-directed spin labeling and ²H-labeled side chains. Pinholin S²¹ is responsible for the permeabilization of the inner cytosolic membrane of double stranded DNA bacteriophage host cells. In this study, we report on the overall global helical structure using circular dichroism (CD) spectroscopy for the active form and the negative-dominant inactive mutant form of S²¹ pinholin. The local helical secondary structure was confirmed for both transmembrane domains (TMDs) for the active and inactive S²¹ pinholin using the ESEEM spectroscopic technique. Comparison of the ESEEM normalized frequency domain intensity for each transmembrane domain gives an insight into the α-helical folding nature of these domains as opposed to a π or 3₁₀-helix which have been observed in other channel forming proteins.     

Genetic Dissection of T4 Lysis

t is the holin gene for coliphage T4, encoding a 218-amino-acid (aa) protein essential for the inner membrane hole formation that initiates lysis and terminates the phage infection cycle. T is predicted to be an integral membrane protein that adopts an Nⁱⁿ-C^out topology with a single transmembrane domain (TMD). This holin topology is different from those of the well-studied holins S105 (3 TMDs; N^out-Cⁱⁿ) of the coliphage lambda and S68 (2 TMDs; Nⁱⁿ-Cⁱⁿ) of the lambdoid phage 21. Here, we used random mutagenesis to construct a library of lysis-defective alleles of t to discern residues and domains important for holin function and for the inhibition of lysis by the T4 antiholin, RI. The results show that mutations in all 3 topological domains (N-terminal cytoplasmic, TMD, and C-terminal periplasmic) can abrogate holin function. Additionally, several lysis-defective alleles in the C-terminal domain are no longer competent in binding RI. Taken together, these results shed light on the roles of the previously uncharacterized N-terminal and C-terminal domains in lysis and its real-time regulation.     

The pinholin of lambdoid phage 21: control of lysis by membrane depolarization

The phage 21 holin, S²¹, forms small membrane holes that depolarize the membrane and is designated as a pinholin, as opposed to large-hole-forming holins, like S^λ. Pinholins require secreted SAR endolysins, a pairing that may represent an intermediate in the evolution of canonical holin-endolysin systems.     

Stable micron‐scale holes are a general feature of canonical holins

At a programmed time in phage infection cycles, canonical holins suddenly trigger to cause lethal damage to the cytoplasmic membrane, resulting in the cessation of respiration and the non‐specific release of pre‐folded, fully active endolysins to the periplasm. For the paradigm holin S105 of lambda, triggering is correlated with the formation of micron‐scale membrane holes, visible as interruptions in the bilayer in cryo‐electron microscopic images and tomographic reconstructions. Here we report that the size distribution of the holes is stable for long periods after triggering. Moreover, early triggering caused by an early lysis allele of S105 formed approximately the same number of holes, but the lesions were significantly smaller. In contrast, early triggering prematurely induced by energy poisons resulted in many fewer visible holes, consistent with previous sizing studies. Importantly, the unrelated canonical holins P2 Y and T4 T were found to cause the formation of holes of approximately the same size and number as for lambda. In contrast, no such lesions were visible after triggering of the pinholin S²¹68. These results generalize the hole formation phenomenon for canonical holins. A model is presented suggesting the unprecedentedly large size of these holes is related to the timing mechanism.     

Assembling an ion channel: ORF 3a from SARS‐CoV

Protein 3a is a 274 amino acid polytopic channel protein with three putative transmembrane domains (TMDs) encoded by severe acute respiratory syndrome corona virus (SARS‐CoV). Synthetic peptides corresponding to each of its three individual transmembrane domains (TMDs) are reconstituted into artificial lipid bilayers. Only TMD2 and TMD3 induce channel activity. Reconstitution of the peptides as TMD1 + TMD3 as well as TMD2 + TMD3 in a 1 : 1 mixture induces membrane activity for both mixtures. In a 1 : 1 : 1 mixture, channel like behavior is almost restored. Expression of full length 3a and reconstitution into artificial lipid bilayers reveal a weak cation selective (P_K ≈ 2 P_Cl) rectifying channel. In the presence of nonphysiological concentration of Ca‐ions the channel develops channel activity. © 2013 Wiley Periodicals, Inc. Biopolymers 99:628–635, 2013.     

Crim1C140S mutant mice reveal the importance of cysteine 140 in the internal region 1 of CRIM1 for its physiological functions

Cysteine-rich transmembrane bone morphogenetic protein regulator 1 (CRIM1) is a type I transmembrane protein involved in the organogenesis of many tissues via its interactions with growth factors including BMP, TGF-β, and VEGF. In this study, we used whole-exome sequencing and linkage analysis to identify a novel Crim1 mutant allele generated by ENU mutagenesis in mice. This allele is a missense mutation that causes a cysteine-to-serine substitution at position 140, and is referred to as Crim1^C140S. In addition to the previously reported phenotypes in Crim1 mutants, Crim1^C140S homozygous mice exhibited several novel phenotypes, including dwarfism, enlarged seminal vesicles, and rectal prolapse. In vitro analyses showed that Crim1^C140S mutation affected the formation of CRIM1 complexes and decreased the amount of the overexpressed CRIM1 proteins in the cell culture supernatants. Cys140 is located in the internal region 1 (IR1) of the N-terminal extracellular region of CRIM1 and resides outside any identified functional domains. Inference of the domain architecture suggested that the Crim1^C140S mutation disturbs an intramolecular disulfide bond in IR1, leading to the protein instability and the functional defects of CRIM1. Crim1^C140S highlights the functional importance of the IR1, and Crim1^C140S mice should serve as a valuable model for investigating the functions of CRIM1 that are unidentified as yet.

     

A Unique Phenylalanine in the Transmembrane Domain Strengthens Homodimerization of the Syndecan-2 Transmembrane Domain and Functionally Regulates Syndecan-2

The syndecans are a type of cell surface adhesion receptor that initiates intracellular signaling events through receptor clustering mediated by their highly conserved transmembrane domains (TMDs). However, the exact function of the syndecan TMD is not yet fully understood. Here, we investigated the specific regulatory role of the syndecan-2 TMD. We found that syndecan-2 mutants in which the TMD had been replaced with that of syndecan-4 were defective in syndecan-2-mediated functions, suggesting that the TMD of syndecan-2 plays one or more specific roles. Interestingly, syndecan-2 has a stronger tendency to form sodium dodecyl sulfate (SDS)-resistant homodimers than syndecan-4. Our structural studies showed that a unique phenylalanine residue (Phe¹⁶⁷) enables an additional molecular interaction between the TMDs of the syndecan-2 homodimer. The presence of Phe¹⁶⁷ was correlated with a higher tendency toward oligomerization, and its replacement with isoleucine significantly reduced the SDS-resistant dimer formation and cellular functions of syndecan-2 (e.g. cell migration). Conversely, replacement of isoleucine with phenylalanine at this position in the syndecan-4 TMD rescued the defects observed in a mutant syndecan-2 harboring the syndecan-4 TMD. Taken together, these data suggest that Phe¹⁶⁷ in the TMD of syndecan-2 endows the protein with specific functions. Our work offers new insights into the signaling mediated by the TMD of syndecan family members.     

Insight into the structures of the second and fifth transmembrane domains of Slc11a1 in membrane mimics

Slc11a1 is an integral membrane protein with 12 putative transmembrane domains and functions as a pH‐coupled divalent metal cation transporter. In the present study, the structures of the peptides corresponding to the second and fifth transmembrane domains of Slc11a1 (from 88 to 109 for TMD2 and from 190 to 215 for TMD5) were determined in membrane‐mimic environments by CD and NMR techniques. It was demonstrated that TMD2 and TMD5 form an α‐helical structure in 30% 2,2,2‐trifluoroethanol (TFE) and 40% hexafluoro‐2‐propanol (HFIP) aqueous solution, respectively. The α‐helix of TMD5 displays a less space‐occupied face consisting of the residues Ala194, Gly197, Thr201, Ala204 and Gly208. The α‐helix is partially unfolded in the N‐terminal region when Gly197 is substituted by Val. The unfolding of the helix in the N‐terminal part and/or increase in volume at the less space‐occupied face of the helix may exert an effect on the arrangement of TMD5 in membrane. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.     

Ultracentrifugation studies on the transmembrane domain of the human erythrocyte anion transporter Band 3 in the detergent C12E8

The dilute solution behaviour of the transmembrane domain (TMD) of the human erythrocyte anion exchanger Band 3 was studied by analytical ultracentrifugation. Sedimentation velocity and equilibrium studies of the TMD solubilized with the detergent C₁₂E₈ demonstrate that the protein is a stable dimer in the concentration range 0.1 to 1 mg/ml. There is no evidence of a dissociation at low concentrations or of an association at higher concentrations. Hydrodynamic calculations applying a prolate ellipsoid of revolution and assuming a hydration of w=0.35 result in an asymmetrical particle with an axial ratio (a/b) of ∼3.5. Received: 8 January 1998 / Revised version: 21 April 1998 / Accepted: 22 April 1998     

Probing the Structure of the S105 Hole

For most phages, holins control the timing of host lysis. During the morphogenesis period of the infection cycle, canonical holins accumulate harmlessly in the cytoplasmic membrane until they suddenly trigger to form lethal lesions called holes. The holes can be visualized by cryo-electron microscopy and tomography as micrometer-scale interruptions in the membrane. To explore the fine structure of the holes formed by the lambda holin, S105, a cysteine-scanning accessibility study was performed. A collection of S105 alleles encoding holins with a single Cys residue in different positions was developed and characterized for lytic function. Based on the ability of 4-acetamido-4′-((iodoacetyl) amino) stilbene-2,2′-disulfonic acid, disodium salt (IASD), to modify these Cys residues, one face of transmembrane domain 1 (TMD1) and TMD3 was judged to face the lumen of the S105 hole. In both cases, the lumen-accessible face was found to correspond to the more hydrophilic face of the two TMDs. Judging by the efficiency of IASD modification, it was concluded that the bulk of the S105 protein molecules were involved in facing the lumen. These results are consistent with a model in which the perimeters of the S105 holes are lined by the holin molecules present at the time of lysis. Moreover, the findings that TMD1 and TMD3 face the lumen, coupled with previous results showing TMD2-TMD2 contacts in the S105 dimer, support a model in which membrane depolarization drives the transition of S105 from homotypic to heterotypic oligomeric interactions.     

A dominant mutation in the bacteriophage lambda S gene causes premature lysis and an absolute defective plating phenotype

The S and R genes of the bacteriophage λ are required for lysis of the host. R encodes ‘endolysin’, a soluble transglycosylase which accumulates in the cytoplasm during late protein synthesis. S encodes a ‘holin’, a small membrane protein which, at a precisely scheduled time, terminates the vegetative cycle by forming a lethal lesion in the membrane through which gpR gains access to the peptidoglycan. A missense allele of S, Ala52Gly, causes lysis to occur prematurely at about 19–20 min after induction of a lysogen, compared to 45min for the wild type. This allele has a severe plaque-forming defect which appears to be entirely a consequence of the early lysis and resultant severe reduction in particle burst size. The early-lysis phenotype is dominant and is aggravated, in terms of an even more reduced burst size, at both 30°C and 42°C. The mutation maps in the middle of a putative membrane-spanning helical domain of S, near the sites of other S⁻ mutations with recessive non-lytic phenotypes. The mutation has no effect on S-protein accumulation or on the ratio of S107 and S105 products in the membrane. The mutation appears to affect the intrinsic timing function by which the S protein controls the lysis schedule.     

Point Mutations in Dimerization Motifs of the Transmembrane Domain Stabilize Active or Inactive State of the EphA2 Receptor Tyrosine Kinase

The EphA2 receptor tyrosine kinase plays a central role in the regulation of cell adhesion and guidance in many human tissues. The activation of EphA2 occurs after proper dimerization/oligomerization in the plasma membrane, which occurs with the participation of extracellular and cytoplasmic domains. Our study revealed that the isolated transmembrane domain (TMD) of EphA2 embedded into the lipid bicelle dimerized via the heptad repeat motif L⁵³⁵X₃G⁵³⁹X₂A⁵⁴²X₃V⁵⁴⁶X₂L⁵⁴⁹ rather than through the alternative glycine zipper motif A⁵³⁶X₃G⁵⁴⁰X₃G⁵⁴⁴ (typical for TMD dimerization in many proteins). To evaluate the significance of TMD interactions for full-length EphA2, we substituted key residues in the heptad repeat motif (HR variant: G539I, A542I, G553I) or in the glycine zipper motif (GZ variant: G540I, G544I) and expressed YFP-tagged EphA2 (WT, HR, and GZ variants) in HEK293T cells. Confocal microscopy revealed a similar distribution of all EphA2-YFP variants in cells. The expression of EphA2-YFP variants and their kinase activity (phosphorylation of Tyr⁵⁸⁸ and/or Tyr⁵⁹⁴) and ephrin-A3 binding were analyzed with flow cytometry on a single cell basis. Activation of any EphA2 variant is found to occur even without ephrin stimulation when the EphA2 content in cells is sufficiently high. Ephrin-A3 binding is not affected in mutant variants. Mutations in the TMD have a significant effect on EphA2 activity. Both ligand-dependent and ligand-independent activities are enhanced for the HR variant and reduced for the GZ variant compared with the WT. These findings allow us to suggest TMD dimerization switching between the heptad repeat and glycine zipper motifs, corresponding to inactive and active receptor states, respectively, as a mechanism underlying EphA2 signal transduction.     

CFTR: Domains, Structure, and Function

Mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR) cause cystic fibrosis (CF) (Collins, 1992). Over 500 naturally occurring mutations have been identified in CF gene which are located in all of the domains of the protein (Kerem et al., 1990; Mercier et al., 1993; Ghanem et al., 1994; Fanen et al., 1992; Ferec et al., 1992; Cutting et al., 1990). Early studies by several investigators characterized CFTR as a chloride channel (Anderson et al.; 1991b,c; Bear et al., 1991). The complex secondary structure of the protein suggested that CFTR might possess other functions in addition to being a chloride channel. Studies have established that the CFTR functions not only as a chloride channel but is indeed a regulator of sodium channels (Stutts et al., 1995), outwardly rectifying chloride channels (ORCC) (Gray et al., 1989; Garber et al., 1992; Egan et al., 1992; Hwang et al., 1989; Schwiebert et al., 1995) and also the transport of ATP (Schwiebert et al., 1995; Reisin et al., 1994). This mini-review deals with the studies which elucidate the functions of the various domains of CFTR, namely the transmembrane domains, TMD1 and TMD2, the two cytoplasmic nucleotide binding domains, NBD1 and NBD2, and the regulatory, R, domain.     

Analysis of Transmembrane Domains 1 and 4 of the Human Angiotensin II AT1 Receptor by Cysteine-scanning Mutagenesis

The octapeptide hormone angiotensin II (AngII) exerts a wide variety of cardiovascular effects through the activation of the AT₁ receptor, which belongs to the G protein-coupled receptor superfamily. Like other G protein-coupled receptors, the AT₁ receptor possesses seven transmembrane domains that provide structural support for the formation of the ligand-binding pocket. Here, we investigated the role of the first and fourth transmembrane domains (TMDs) in the formation of the binding pocket of the human AT₁ receptor using the substituted-cysteine accessibility method. Each residue within the Phe-28^(1.32)–Ile-53^(1.57) fragment of TMD1 and Leu-143^(4.40)–Phe-170^(4.67) fragment of TMD4 was mutated, one at a time, to a cysteine. The resulting mutant receptors were expressed in COS-7 cells, which were subsequently treated with the charged sulfhydryl-specific alkylating agent methanethiosulfonate ethylammonium (MTSEA). This treatment led to a significant reduction in the binding affinity of TMD1 mutants M30C^(1.34)-AT₁ and T33C^(1.37)-AT₁ and TMD4 mutant V169C^(4.66)-AT₁. Although this reduction in binding of the TMD1 mutants was maintained when examined in a constitutively active receptor (N111G-AT₁) background, we found that V169C^(4.66)-AT₁ remained unaffected when treated with MTSEA compared with untreated in this context. Moreover, the complete loss of binding observed for R167C^(4.64)-AT₁ was restored upon treatment with MTSEA. Our results suggest that the extracellular portion of TMD1, particularly residues Met-30^(1.34) and Thr-33^(1.37), as well as residues Arg-167^(4.64) and Val-169^(4.66) at the junction of TMD4 and the second extracellular loop, are important binding determinants within the AT₁ receptor binding pocket but that these TMDs undergo very little movement, if at all, during the activation process.     

The dimerization of PSGL‐1 is driven by the transmembrane domain via a leucine zipper motif

P‐selectin glycoprotein ligand‐1 (PSGL‐1) is a homodimeric mucin ligand that is important to mediate the earliest adhesive event during an inflammatory response by rapidly forming and dissociating the selectin‐ligand adhesive bonds. Recent research indicates that the noncovalent associations between the PSGL‐1 transmembrane domains (TMDs) can substitute for the C320‐dependent covalent bond to mediate the dimerization of PSGL‐1. In this article, we combined TOXCAT assays and molecular dynamics (MD) simulations to probe the mechanism of PSGL‐1 dimerization. The results of TOXCAT assays and Martini coarse‐grained molecular dynamics (CG MD) simulations demonstrated that PSGL‐1 TMDs strongly dimerized in a natural membrane and a leucine zipper motif was responsible for the noncovalent dimerization of PSGL‐1 TMD since mutations of the residues that occupied a or d positions in an (abcdefg)n leucine heptad repeat motif significantly reduced the dimer activity. Furthermore, we studied the effects of the disulfide bond on the PSGL‐1 dimer using MD simulations. The disulfide bond was critical to form the leucine zipper structure, by which the disulfide bond further improved the stability of the PSGL‐1 dimer. These findings provide insights to understand the transmembrane association of PSGL‐1 that is an important structural basis for PSGL‐1 preferentially binding to P‐selectin to achieve its biochemical and biophysical functions.     

Genetic analysis of ryanodine receptor function in<Emphasis Type="Italic"> Caenorhabditis elegans</Emphasis> based on<Emphasis Type="Italic"> unc-68</Emphasis> revertants

The Caenorhabditis elegans ryanodine receptor is encoded by the unc-68 gene, and functions as a Ca²⁺-induced Ca²⁺ release channel during muscle contraction. To investigate the factors that suppress calcium release and identify molecules that interact with the ryanodine receptor, we isolated revertants from two unc-68 mutants. Three of the revertants obtained from the null allele unc-68(e540), which displayed normal motility, had intragenic mutations that resulted in failure to splice out intron 21. The other two, kh53 and kh55, had amino acid insertions in the third of the four RyR domains. The brood size and the egg laying rate remain abnormal in these revertants. This suggests the third RyR domain may be required for egg laying and embryogenesis, although we can not determine a molecular mechanism. Five ketamine sensitive revertants recovered from the missense mutant unc-68(kh30) showed altered responses to caffeine, ryanodine, levamisole and ouabain relative to those of the unc-68(kh30) animals. These may carry second-site suppressor mutations, which may define genes for proteins that regulate the Ca²⁺ concentration in body-wall muscle. One of these mutants, kh52 , shows lower motility and higher sensitivity to drugs, and this mutation was mapped to chromosome X. These observations provide a basis for the study of ryanodine receptor functions in embryogenesis and in calcium-mediated regulation of muscle contraction in C. elegans. This is the first study to show that the conserved RyR domain of the receptor acts in egg laying and embryogenesis.Communicated by C. P. Hollenberg     

Catalase polymorphism in the red cells of horses*

Catalase zymograms performed on hemolysates of horse red cells revealed three phenotypes each composed of a single zone of activity designated F, M and S in decreasing order of migration towards the anode. It is proposed that these phenotypes are controlled by a pair of alleles, Cat^F and Cat^S, such that the genotypes Cat^FCat^F, Cat^FCat^S and Cat^SCat^S give rise respectively to phenotypes F, M and S. The effects of storage, heat, UV light and selected reagents on these phenotypes are discussed.