lördag 18 juli 2009

Modeling at the Gates of the Cell

Got a mail about possibility to win 25000 dollars and there it was. The model of a gap junction. Last year Dr Fleischman received such a prize. He investigated the structure, function, and evolution of membrane proteins associated with hereditary hearing loss and neurodegenerative diseases, cancer, and bacterial drug resistance. He ties to understand the mechanistic relationship between molecular structure and function in human health and disease.

He says: Lack of an atomic resolution structure of the gap junction has made it extremely difficult to conduct biochemical investigations within a consistent framework ... it becomes clear that we have an unsatisfactory picture of the possible structural motifs found in membrane proteins. This limited viewpoint raises the question: Can we apply computational modeling to provide additional insight into the relationship between structure and function in membrane proteins? Experimental data along with evolutionary analysis may help us bridge the gap in our structural understanding of membrane proteins and provide structural models.

An updated overview of current knowledge of connexins and their interacting proteins and connexin modulation, disease and tumorigenesis is made by Dbouk et.al. 2009.

The cores of membrane proteins are much more evolutionarily conserved than their peripheries. The reason for this is simple: Mutation at the core of the protein is much more likely to disrupt the protein structure than one in a lipid-facing position, and would be eliminated by the forces of natural selection. Evolutionary conservation could therefore distinguish the parts of each helix that face the lipid from those that face the core of the protein.

Gap junctions links the cytoplasms of neighboring cells in mammalian tissues and allows the cells to transfer metabolites and signals. It is a critical component in cellular signaling of many tissues, and numerous mutations in its membrane domain have been implicated in hereditary hearing loss, neurodegenerative disease, and other genetic diseases.

Molecular organization of a recombinant gap junction channel. The approximate boundaries for the membrane bilayers (M), extracellular gap (E), and the cytoplasmic space (C) are indicated.

(a) A top view looking toward the extracellular gap and (b) a side view of part of the 3D map of a recombinant gap junction channel. The 24 well-resolved rod-shaped features reveal the packing of the transmembrane alpha helices, and four have been arbitrarily labeled A, B, C, and D.

Gap junction channels are formed by the end-to-end docking of two hemichannels or connexons = hexamers, from adjacent membranes, each of which displayed 24 rods of density in the membrane interior. Each connexon comprises six connexin subunits, proteins which are encoded by ~20 isoforms in the human genome. All connexins contain four transmembrane (TM)4 segments (M1-M4), whose N and C termini are located in the cytoplasm. The extracellular aspect of the hemichannel is composed of two extracellular loops (E1 and E2) from each connexin monomer. A long-awaited high-resolution structure of a connexin channel was published recently (Maeda et al. 2009).

The gating mechanism that achieves opening is voltage and pH sensitive, and protein phosphorylation - sensitive, and requires extracellular calcium ion in the millimolar range to remain closed at normal resting potentials. Unapposed connexin hemichannels exhibit robust closure in response to membrane hyperpolarization and extracellular calcium. This form of gating, termed "loop gating," is largely responsible for regulating hemichannel opening. The molecular components and structural rearrangements underlying loop gating remain unknown. Metal bridges can lock up the gate. Sulfhydryl groups, the contribution from disulfide bond formation and other residues that coordinate metal ions with high affinity are other candidates. Metal ions access the cysteine side chains through the open pore and that closure of the loop gate involves movement of the TM1/E1 region that results in local narrowing of the large aqueous connexin pore. The loop gate must also be able to open when docked to another hemichannel in the junctional configuration. The relationship between loop gating and the mechanism/structure of hemichannel docking is unclear. Perhaps the mechanism of loop gate formation is different in connexin isoforms? An alternative possibility is that the binding and the opening are two distinct processes that are not obligatorily linked. This scenario permits docking without opening of the pore; docking would enable opening but not require it. In this case, the structural elements involved in the two processes would need to be distinct.

What about the basis of the voltage sensitivity of the loop gate? Connexin channels have at least one other gating mechanism, known as Vj gating, which closes the channels to a substate and is well characterized at the single-channel level. The sensor for this type of gating was within the pore, composed of the amino-terminal domain of the protein, which when the gate is open is folded into the lumen of pore against TM1, forming the pore wall in the cytoplasmic end of the pore. The suggestion is that in response to an appropriate electric field, these domains peel off the pore wall and move toward the cytoplasm to collapse into an aggregate that largely occludes the lumen. This is a unique voltage-dependent gating mechanism, operating at the opposite end of the pore from the loop gate. Data suggest that the voltage sensors of the two mechanisms are in series in the lumen of the pore, and that the sensitivity of each to applied voltage changes with the position of the other gate.

The cytoplasmic N-termini of connexins have been implicated in protein trafficking, oligomerization and channel gating. Mutants containing nine or more N-terminal amino acids form gap junction plaques. This N-terminal peptide is predominantly {alpha}-helical. The {alpha}-helical structure of the connexin37 N terminus may be dispensable for protein localization, but it is required for channel and hemichannel function. As much as half the length of the connexin N-terminus can be deleted without affecting formation of gap junction plaques, but an intact N-terminus is required for hemichannel gating and intercellular communication. The NT does not need to be the gate itself. Loss of conductance in the NT deletion mutants might also result if an intact NT is required for the other gates to be operative.

Superposition of cross sections (red and blue) within the hydrophobic region of the two hexameric connexons forming the gap junction channel. The shaded regions identify three possible boundaries for the connexin subunit. With reference to the labels in Fig. 3a, the models show (a) a bundle of four alpha helices (A'DCB'), (b) a check-mark arrangement (ABCD), and (c) a zigzag pattern (BCDA'). White arrows identify axes of symmetry that are in plane, noncrystallographic, and twofold.

The model structure of the gap junction membrane domain helps to explain the differential effect of disease-causing and benign polymorphisms.

Fleishmans group made a model of a gap junction domain. The model structure helped identify positions on adjacent helices where second-site mutations restored membrane localization, revealing possible interactions between residue pairs. We thus identified two putative salt bridges and one pair involved in packing interactions in which one disease-causing mutation suppressed the effects of another. These results seem to reveal interactions that apparently stabilize contacts between TM helices in connexins and suggest that abrogation of such interactions bring about some of the effects of disease-causing mutations.

The stabilizing structure is a triad of charged positions (salt-bridges), the first two occupied by basic residues and the last by an acidic residue, are highly conserved throughout all connexins. In theory, the Arg and Glu residues, which are reciprocally charged and are near the water-filled pore lumen, could be involved in stabilizing electrostatic interactions; it has been estimated that salt bridges embedded in water can add nearly 1 kcal/mol to protein stability. Additional forces contribute to stabilization (no salt bridge).

Although wild-type connexins are membrane-localized, our images show fluorescence also outside the membrane, even in wild-type connexin. Localization of wild-type connexins outside the membrane, in addition to the existence of gap junction plaques, have been observed in other studies involving overexpressed connexins, and it has been suggested that the cytoplasmic fraction of the protein is at least in part localized in aggresomes. The important point to notice is that, along with the localization of some of the protein in the cytoplasm, wild-type and doubly mutated connexins are localized in the plasma membrane, whereas the single mutants are not.

Tight junctions
Unger et.al. write:... the protein density that formed the extracellular vestibule provided a tight seal to exclude the exchange of substances with the extracellular milieu.

Remembers me of tight junctions. In brain we have the brainbarrier, in nerves we have a tight junction seal, in liver and heart too. What kind of purpose do that have? Sorting big molecules out only?

From the rewiev: The adherens and tight junctions regulates paracellular permeability (barrier function) and cell polarity, among other functions. Gap junctions may even regulate the expression and function of tight junction proteins. The tight junction membrane-associated guanylate kinase protein is involved in the organization and trafficking of gap junctions, and mediates the delivery of Cx43 from a lipid raft domain to gap junctional plaques. Cx32 can participate in the formation of functional tight junctions and in actin organization. Interactions of connexins with the actin cytoskeleton and associated proteins serve to stabilize gap junctions at the plasma membrane.

Among newly-discovered interacting connexin partners, plasma membrane ion channels, membrane transport proteins and receptors have been shown to interact directly or indirectly with connexins, and these include aquaporin-0 and acetylcholine receptors, among others. The calcium/calmodulin-dependant kinase II (CaMKII) interacts with and phosphorylates. Other interactions include cholesterol, COX-2 and heat shock protein 90 (Hsp90) and translocase of the outer mitochondrial membrane.

Interaction of connexins with CaMKII may have a general regulatory role in neuronal signal transmission, with a role in electrical coupling in addition to the defined role of CaMKII in chemical synaptic transmission.

Recent studies have revealed complex translational and post-translational mechanisms that regulate connexin synthesis, maturation, membrane transport and degradation that in turn modulate gap junction intercellular communication. With the growing myriad of connexin interacting proteins, including cytoskeletal elements, junctional proteins, and enzymes, gap junctions are now perceived, not only as channels between neighboring cells, but as signaling complexes that regulate cell function and transformation. They exert their effects on proliferation and other aspects of life and death of the cell through mostly-undefined mechanisms.

An array of studies illustrated the important contribution of GJIC to developmental and regulatory events such as embryonic growth, bone modeling, alveolar differentiation, central nervous system signaling and neural function in the developing central nervous system, also in maintenance of tissue homeostasis through processes such as synchronization.

Tis is very interesting for my meridians. Very, very much.

Sarel J. Fleishman, Adi D. Sabag, Eran Ophir, Karen B. Avraham, and Nir Ben-Tal 2006: The Structural Context of Disease-causing Mutations in Gap Junctions. J. Biol. Chem., Vol. 281, Issue 39, 28958-28963, September 29, 2006. http://www.jbc.org/cgi/content/full/281/39/28958

Sarel Fleishman 2008: Modeling at the Gates of the Cell.

Vinzenz M. Unger, Nalin M. Kumar, Norton B. Gilula, Mark Yeager 1999: Three-Dimensional Structure of a Recombinant Gap Junction Membrane Channel. Science 19 February 1999: Vol. 283. no. 5405, pp. 1176 - 1180. http://www.sciencemag.org/cgi/content/full/283/5405/1176

A. L. Harris, 2009: Gating on the outside. J. Gen. Physiol., June 1, 2009; 133(6): 549 - 553. http://jgp.rupress.org/cgi/content/full/133/6/549

Maeda, S., S. Nakagawa, M. Suga, E. Yamashita, A. Oshima, Y. Fujiyoshi, and T. Tsukihara. 2009. Structure of the connexin 26 gap junction channel at 3.5 A resolution. Nature. 458:597–602.

Oshima, A., Doi, T., Mitsuoka, K., Maeda, S. and Fujiyoshi, Y. (2003). Roles of Met-34, Cys-64, and Arg-75 in the assembly of human connexin 26: Implication for key amino acid residues for channel formation and function. J. Biol. Chem. 278, 1807-1816. http://www.jbc.org/cgi/content/abstract/278/3/1807?ijkey=7588b0090e287999b2f3cc079c2c694006d66731&keytype2=tf_ipsecsha

J. W. Kyle, P. J. Minogue, B. C. Thomas, D. A. L. Domowicz, V. M. Berthoud, D. A. Hanck, and E. C. Beyer (2008): An intact connexin N-terminus is required for function but not gap junction formation. J. Cell Sci. 121, 2744-2750 http://jcs.biologists.org/cgi/content/full/121/16/2744

V. K. Verselis, M. P. Trelles, C. Rubinos, T. A. Bargiello, and M. Srinivas (2009): Loop Gating of Connexin Hemichannels Involves Movement of Pore-lining Residues in the First Extracellular Loop Domain. J. Biol. Chem. 284, 4484-4493.

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