Peripherin-2: an intracellular analogy to viral fusion proteins.

The C-terminus of the intracellular retinal rod outer segment disk protein peripherin-2 binds to membranes, adopts a helical conformation, and promotes membrane fusion, which suggests an analogy to the structure and function of viral envelope fusion proteins. Nuclear magnetic resonance (NMR) data and fluorescence data show that a 63-residue polypeptide comprising the C-terminus of bovine peripherin-2 (R284-G346) binds to the membrane mimetic, dodecylphosphocholine micelles. High-resolution NMR studies reveal that although this C-terminal fragment is unstructured in solution, the same fragment adopts helical structure when bound to the micelles. The C-terminus may be a member of the class of intrinsically unstructured protein domains. Using methods developed for the G-protein coupled receptor rhodopsin, a model for the structure of the transmembrane domain of peripherin-2 was constructed. Previously published data showed that both peripherin-2 and viral fusion proteins are transmembrane proteins that promote membrane fusion and have a fusion peptide sequence within the protein that independently promotes membrane fusion. Furthermore, the fusion-active sequence of peripherin-2 exhibits a sequence motif that matches the viral fusion peptide of influenza hemagglutinin (HA). These observations collectively suggest that the mechanism of intracellular membrane fusion induced by peripherin-2 and the mechanism of enveloped viral fusion may have features in common.

Calcium-dependent association of calmodulin with the C-terminal domain of the tetraspanin protein peripherin/rds.

Peripherin/rds (p/rds), an integral membrane protein from the transmembrane 4 (TMF4) superfamily, possesses a multi-functional C-terminal domain that plays crucial roles in rod outer segment (ROS) disk renewal and structure. Here, we report that the calcium binding protein calmodulin (CaM) binds to the C-terminal domain of p/rds. Fluorescence spectroscopy reveals Ca2+-dependent association of CaM with a polypeptide corresponding to the C-terminal domain of p/rds. The fluorescence anisotropy of the polypeptide upon CaM titration yields a dissociation constant (KD) of 320 +/- 150 nM. The results of the fluorescence experiments were confirmed by GST-pull down analyses in which a GST-p/rds C-terminal domain fusion protein was shown to pull down CaM in a calcium-dependent manner. Moreover, molecular modeling and sequence predictions suggest that the CaM binding domain resides in a p/rds functional hot spot, between residues E314 and G329. Predictions were confirmed by peptide competition studies and a GST-p/rds C-terminal domain construct in which the putative Ca2+/CaM binding site was scrambled. This GST-polypeptide did not associate with Ca2+/CaM. This putative calmodulin domain is highly conserved between human, mouse, rat, and bovine p/rds. Finally, the binding of Ca2+/CaM inhibited fusion between ROS disk and ROS plasma membranes as well as p/rds C-terminal-domain-induced fusion in model membrane studies. These results offer a new mechanism for the modulation of p/rds function.

Structural studies of the putative helix 8 in the human beta(2) adrenergic receptor: an NMR study.

The recently reported crystal structure of bovine rhodopsin revealed a cytoplasmic helix (helix 8) in addition to the seven transmembrane helices. This domain is roughly perpendicular to the transmembrane bundle in the presence of an interface and may be a loop-like structure in the absence of an interface. Several studies carried out on this domain suggested that it might act as a conformational switch between the inactive and activated states of this G-protein coupled receptor (GPCR). These results raised the question whether helix 8 may be an important feature of other GPCRs as well. To explore this question, we determined the structure of a peptide representing the putative helix 8 domain in another receptor that belongs to the rhodopsin family of GPCRs, the human beta(2) adrenergic receptor (hbeta(2)AR), using two-dimensional (1)H nuclear magnetic resonance (NMR). The key results from this structural study are that the putative helix 8 domain is helical in detergent and in DMSO while in water this region is disordered; the conformation is therefore dependent upon the environment. Comparison of data from five GPCRs suggests that these observations may be generally important for GPCR structure and function.

Studies on the structure of the G-protein-coupled receptor rhodopsin including the putative G-protein binding site in unactivated and activated forms.

Activation of G-protein coupled receptors (GPCR) is not yet understood. A recent structure showed most of rhodopsin in the ground (not activated) state of the GPCR, but the cytoplasmic face, which couples to the G protein in signal transduction, was not well-defined. We have determined an experimental three-dimensional structure for rhodopsin in the unactivated state, which shows good agreement with the crystal structure in the transmembrane domain. This new structure defines the cytoplasmic face of rhodopsin. The G-protein binding site can be mapped. The same experimental approach yields a preliminary structure of the cytoplasmic face in the activated (metarhodopsin II) receptor. Differences between the two structures suggest how the receptor is activated to couple with transducin.

Structures of the transmembrane helices of the G-protein coupled receptor, rhodopsin.

An hypothesis is tested that individual peptides corresponding to the transmembrane helices of the membrane protein, rhodopsin, would form helices in solution similar to those in the native protein. Peptides containing the sequences of helices 1, 4 and 5 of rhodopsin were synthesized. Two peptides, with overlapping sequences at their termini, were synthesized to cover each of the helices. The peptides from helix 1 and helix 4 were helical throughout most of their length. The N- and C-termini of all the peptides were disordered and proline caused opening of the helical structure in both helix 1 and helix 4. The peptides from helix 5 were helical in the middle segment of each peptide, with larger disordered regions in the N- and C-termini than for helices 1 and 4. These observations show that there is a strong helical propensity in the amino acid sequences corresponding to the transmembrane domain of this G-protein coupled receptor. In the case of the peptides from helix 4, it was possible to superimpose the structures of the overlapping sequences to produce a construct covering the whole of the sequence of helix 4 of rhodopsin. As similar superposition for the peptides from helix 1 also produced a construct, but somewhat less successfully because of the disordering in the region of sequence overlap. This latter problem was more severe for helix 5 and therefore a single peptide was synthesized for the entire sequence of this helix, and its structure determined. It proved to be helical throughout. Comparison of all these structures with the recent crystal structure of rhodopsin revealed that the peptide structures mimicked the structures seen in the whole protein. Thus similar studies of peptides may provide useful information on the secondary structure of other transmembrane proteins built around helical bundles.

Assembly of a polytopic membrane protein structure from the solution structures of overlapping peptide fragments of bacteriorhodopsin.

Three-dimensional structures of only a handful of membrane proteins have been solved, in contrast to the thousands of structures of water-soluble proteins. Difficulties in crystallization have inhibited the determination of the three-dimensional structure of membrane proteins by x-ray crystallography and have spotlighted the critical need for alternative approaches to membrane protein structure. A new approach to the three-dimensional structure of membrane proteins has been developed and tested on the integral membrane protein, bacteriorhodopsin, the crystal structure of which had previously been determined. An overlapping series of 13 peptides, spanning the entire sequence of bacteriorhodopsin, was synthesized, and the structures of these peptides were determined by NMR in dimethylsulfoxide solution. These structures were assembled into a three-dimensional construct by superimposing the overlapping sequences at the ends of each peptide. Onto this construct were written all the distance and angle constraints obtained from the individual solution structures along with a limited number of experimental inter-helical distance constraints, and the construct was subjected to simulated annealing. A three-dimensional structure, determined exclusively by the experimental constraints, emerged that was similar to the crystal structure of this protein. This result suggests an alternative approach to the acquisition of structural information for membrane proteins consisting of helical bundles.

Three dimensional structure of the seventh transmembrane helical domain of the G-protein receptor, rhodopsin.

PURPOSE: The three dimensional structure of a peptide comprising the sequence of the seventh transmembrane segment of the G-protein coupled receptor, rhodopsin, was determined in solution. METHODS: The sequence of the seventh transmembrane segment of rhodopsin, which contains the NPxxY sequence that is highly conserved among G-protein coupled receptors and lys296 that forms the Schiff base with the retinal, was synthesized by solid phase peptide synthesis. The three dimensional structure was determined in solution by high-resolution nuclear magnetic resonance (NMR). RESULTS: The structure revealed a helix-break-helix motif for this sequence. Two families of structures were observed which differed in the angle between the two helical segments. The sequence of this transmembrane segment overlapped significantly the sequence of a peptide from the carboxyl terminal of rhodopsin, the structure of which was solved previously. The redundant sequence formed a helix in both peptides. It was therefore possible to superimpose the redundant sequence of both peptides and construct a structure for rhodopsin encompassing residues 291-348. CONCLUSIONS: This structure reveals locations of the lys296 and the acylation sites of rhodopsin that are consistent with the known biochemistry of this receptor. This segmentation approach to membrane protein structure provides important structural information in the absence of an X-ray crystal structure of rhodopsin. The approach is expected to be useful for other G-protein coupled receptors.

Structures of the intradiskal loops and amino terminus of the G-protein receptor, rhodopsin.

The intradiskal surface of the transmembrane protein, rhodopsin, consists of the amino terminal domain and three loops connecting six of the seven transmembrane helices. This surface corresponds to the extracellular surface of other G-protein receptors. Peptides that represent each of the extramembraneous domains on this surface (three loops and the amino terminus) were synthesized. These peptides also included residues which, based on a hydrophobic plot, could be expected to be part of the transmembrane helix. The structure of each of these peptides in solution was then determined using two-dimensional 1H nuclear magnetic resonance. All peptide domains showed ordered structures in solution. The structures of each of the peptides from intradiskal loops of rhodopsin exhibited a turn in the central region of the peptide. The ends of the peptides show an unwinding of the transmembrane helices to form this turn. The amino terminal domain peptide exhibited alpha-helical regions with breaks and bends at proline residues. This region forms a compact domain. Together, the structures for the loop and amino terminus domains indicate that the intradiskal surface of rhodopsin is ordered. These data further suggest a structural motif for short loops in transmembrane proteins. The ordered structures of these loops, in the absence of the transmembrane helices, indicate that the primary sequences of these loops are sufficient to code for the turn.

Solution structure of the loops of bacteriorhodopsin closely resembles the crystal structure.

Bacteriorhodopsin is one of very few transmembrane proteins for which high resolution structures have been solved. The structure shows a bundle of seven helices connected by six turns. Some turns in proteins are stabilized by short range interactions and can behave as small domains. These observations suggest that peptides containing the sequence of the turns in a membrane protein such as bacteriorhodopsin may form stable turn structures in solution. To test this hypothesis, we determined the solution structure of three peptides each containing the sequence of one of the turns in bacteriorhodopsin. The solution structures of the peptides closely resemble the structures of the corresponding turns in the high resolution structures of the intact protein.

Solution structure of the sixth transmembrane helix of the G-protein-coupled receptor, rhodopsin.

Low resolution electron density maps have revealed the general orientation of the transmembrane helices of rhodopsin. However, high resolution structural information for the transmembrane domain of the G-protein-coupled receptor, rhodopsin, is as yet unavailable. In this study, a high resolution solution structure is reported for a 15 residue portion of the sixth transmembrane helix of rhodopsin (rhovih) as a free peptide. Helix 6 is one of the transmembrane helices of rhodopsin that contains a proline (amino acid residue 267) and the influence of this proline on the structure of this transmembrane domain was unknown. The structure obtained shows an alpha-helix through most of the sequence. The proline apparently induces only a modest distortion in the helix. Previously, the structure of the intradiskal loop connected to helix 6 was solved. The sequence of this loop contained five residues in common (residues 268-272) with the peptide reported here from the rhovih. The five residues in common between these two structures were superimposed to connect these two structures. The superposition showed a root mean square deviation of 0.2 A. Thus, this five residue sequence formed the same structure in both peptides, indicating that the structure of this region is governed primarily by short range interactions.

Altering the state of phosphorylation of rat liver keratin intermediate filaments by ethanol treatment in vivo changes their structure.

Dephosphorylation of keratin intermediate filaments (IF) in livers from ethanol-fed rats relative to controls occurs concurrently with a reorganization of the distribution of IF in the cells. One possible molecular mechanism for this reorganization is a phosphorylation-induced conformational change in the keratin that propagates as a change in the polymerization of the keratin subunits. To test this hypothesis, the structure of liver keratin IF, from both control and alcohol-fed rats, was explored by circular dichroism (CD), tryptophan fluorescence quenching, and 13C nuclear magnetic resonance (NMR). Keratin IF were isolated from livers of control rats and from livers of rats that had ethanol included in their feed for 6-40 weeks. A significant decrease in the intensity of the CD spectrum of keratin IF from livers of ethanol-treated animals, relative to controls, was observed. These data suggested either that a change in conformation or an increase in conformational motility in the keratin IF from ethanol-treated animals occurred as a result of the ethanol-induced dephosphorylation. 13C NMR data were obtained to distinguish between these two possibilities. An increase in resonance intensity of some 13C NMR resonances was observed in the keratin IF from livers of ethanol-treated animals, relative to controls. The CD and NMR data were therefore consistent with an increase in conformational motility of the rod domain in these keratin IF. No significant change was observed in the quenching of tryptophan fluorescence by KI. The change in protein dynamics detected in these experiments could be the molecular basis for the alteration of keratin IF organization in alcoholic hepatitis.

Effects of phosphorylation on the structure of the G-protein receptor rhodopsin.

Upon activation by light, rhodopsin is subject to phosphorylation by rhodopsin kinase at serine and threonine residues in the carboxyl terminal region of the protein. A 19 amino acid peptide that corresponds to the carboxyl terminal end of rhodopsin (residues 330-348) and contains these phosphorylation sites was synthesized. The structure of this peptide was determined using two-dimensional proton NMR. The structure of this peptide was similar to that determined for this region in peptides corresponding to the carboxyl 33 and 43 amino acids of rhodopsin. The effect of phosphorylation on the structure of the carboxyl terminal domain of rhodopsin was determined by solving the solution structures of the peptide containing residues 330-348 with phosphorylation at one (residue 343), three (residues 343, 338, and 334) and seven residues (residues 334, 335, 336, 338, 340, 342, 343). These data indicate that the major structural change occurs upon phosphorylation of the first residue, and that an additional structural change occurs with seven phosphates.

Three-dimensional structure of the cytoplasmic face of the G protein receptor rhodopsin.

Rhodopsin is a G protein receptor from a many-membered family of membrane receptors. No high-resolution structure exists for any member of this family due to the insolubility of membrane proteins and the difficulty in crystallizing membrane proteins. Two new approaches to the structure of rhodopsin are described that circumvent these limitations: (1) individual solution structures of the four cytoplasmic domains of rhodopsin are fitted with the transmembrane domain; (2) the solution structure of a complex of the four cytoplasmic domains is determined from nuclear magnetic resonance data. The two structures are similar. To test the validity of these structures, specific site-to-site distances measured on intact membrane-bound rhodopsin are compared to the same distances on the structures reported here. Excellent agreement is obtained. Furthermore, the agreement is obtained with distances measured on the activated form of teh receptor and not with distances on the dark-adapted form of rhodopsin. This approach may prove to have general applicability for the determination of the structure for membrane proteins.

A distance measurement between specific sites on the cytoplasmic surface of bovine rhodopsin in rod outer segment disk membranes.

Structural information on mammalian integral membrane proteins is scarce. As part of work on an alternative approach to the structure of bovine rhodopsin, a method was devised to obtain an intramolecular distance between two specific sites on rhodopsin while in the rod outer segment disk membrane. In this report, the distance between the rhodopsin kinase phosphorylation site(s) on the carboxyl terminal and the top of the third transmembrane helix was measured on native rhodopsin. Rhodopsin was labeled with a nuclear spin label (31P) by limited phosphorylation with rhodopsin kinase. Major phosphorylation occurs at serines 343 and 338 on the carboxyl terminal. The phosphorylated rhodopsin was then specifically labeled on cysteine 140 with an electron spin label. Magic angle spinning 31P-nuclear magnetic resonance revealed the resonance arising from the phosphorylated protein. The enhancement of the transverse relaxation of this resonance by the paramagnetic spin label was observed. The strength of this perturbation was used to determine the through-space distance between the phosphorylation site(s) and the spin label position. A distance of 18 +/- 3 A was obtained.

The first and second cytoplasmic loops of the G-protein receptor, rhodopsin, independently form beta-turns.

The cytoplasmic face of the transmembrane protein, rhodopsin, is made up of one carboxyl terminal and three cytoplasmic loops connecting six of the seven transmembrane helices. Neither the high-resolution, three-dimensional structure of this G-protein receptor nor any other cell surface receptor is known. In this work, the structures of peptides containing the amino acid sequence of the first and second cytoplasmic loops of rhodopsin have been determined. Both loops show ordered structures in solution. In both loops, the ends of the transmembrane helices unwind and form a beta-turn. The conformations of the two loops are remarkably similar, even though their sequences are not. These data suggest a structural motif for short loops in transmembrane proteins. The well-ordered structures of these loops, in the absence of the transmembrane helices, indicate that the primary sequences of these loops stabilize the beta-turn. These data further suggest that the loops may contribute to the folding of such membrane proteins during their synthesis and insertion into membranes.

Structure determination of the fourth cytoplasmic loop and carboxyl terminal domain of bovine rhodopsin.

PURPOSE: High resolution structural information is lacking for any member of the class of G-protein receptors. This dearth of structural information extends to virtually all integral membrane proteins. As part of an alternative approach to examining integral membrane protein structure, we are determining the structures of the extramembraneous domains of the G-protein receptor, rhodopsin. METHODS: The carboxyl terminal domain of bovine rhodopsin was synthesized, containing the last 43 amino acids of the protein sequence (rhoIVe). This sequence included the entire putative fourth cytoplasmic loop as well as a significant portion of helix seven, the transmembrane helix of this receptor to which the carboxyl terminal is attached. The solution structure of rhoIVe was determined by multidimensional 1H nuclear magnetic resonance. RESULTS: The structure contained a portion of alpha-helix corresponding to the top of transmembrane helix seven of the receptor. This allowed unambiguous docking of the carboxyl terminal domain to a model of the transmembrane domain. Helix seven is longer than suggested by hydropathy analysis. The structure also revealed the fourth cytoplasmic loop. The palmitoylation sites of rhodopsin are located near the deduced membrane surface. However, palmitoylation is not required for formation of this loop. CONCLUSIONS: The carboxyl terminal of rhodopsin forms a structural domain whose structure can be determined separately from the rest of the protein. This structure reveals the fourth cytoplasmic loop that had been suggested to exist based on the presence of palmitoylation sites in the carboxyl terminal domain. Determination of the structure of all of the cytoplasmic domains of rhodopsin in a manner that allows docking to the structure of the transmembrane domain should permit construction of the entire surface of rhodopsin that interacts with the G-protein, transducin. Additionally, the rhodopsin phosphorylation sites and mutations associated with certain autosomal dominant forms of retinitis pigmentosa can now be located in the three dimensional structure of the carboxyl terminal domain.

Rhodopsin-cholesterol interactions in bovine rod outer segment disk membranes.

Cholesterol modulates the function of rhodopsin in the retinal rod outer segment (ROS) disk membranes. One mechanism for such modulation is cholesterol modulation of the properties of the membrane bilayer. This has been explored previously. Another possible mechanism is an interaction between the sterol and the protein, which has not been previously explored. In this study, the fluorescent sterol, cholestatrienol, was used to probe interactions between cholesterol and rhodopsin in bovine ROS disk membranes. Cholestatrienol was incorporated into the disk membranes by exchange from donor phospholipid vesicles. Fluorescence energy transfer from protein tryptophans to cholestatrienol was observed indicating close approach of this fluorescent sterol to the tryptophan. The effectiveness of the energy transfer was measured by the quenching of tryptophan fluorescence by cholestatrienol. The quenching of tryptophan fluorescence was directly related to the cholestatrienol content of the membranes. Cholesterol was incorporated into the disk membranes by exchange from donor phospholipid vesicles. The effect of increasing membrane cholesterol on the ability of cholestatrienol to quench rhodopsin tryptophan fluorescence was determined. This quenching was inversely proportional to the membrane cholesterol content. Furthermore the observed quenching was greater than could be explained by a simple dilution of the cholestatrienol by the addition of cholesterol to the membrane. These data suggest an interaction between the sterol and the protein. The specificity of this interaction was explored by the addition of ergosterol, instead of cholesterol, to the disk membranes. Ergosterol was not able to inhibit the quenching of protein trytophans beyond that due to dilution of the cholestatrienol by addition of ergosterol to the membrane. The ability of cholesterol to compete with cholestatrienol for that interaction suggests a 'site' at which cholesterol contacts rhodopsin. The inability of ergosterol to compete with cholestatrienol for this 'site' suggested that the site was specific for the structure of cholesterol.