Accessing gap-junction channel structure-function relationships through molecular modeling and simulations

Background

Gap junction channels (GJCs) are massive protein channels connecting the cytoplasm of adjacent cells. These channels allow intercellular transfer of molecules up to ~1 kDa, including water, ions and other metabolites. Unveiling structure-function relationships coded into the molecular architecture of these channels is necessary to gain insight on their vast biological function including electrical synapse, inflammation, development and tissular homeostasis. From early works, computational methods have been critical to analyze and interpret experimental observations. Upon the availability of crystallographic structures, molecular modeling and simulations have become a valuable tool to assess structure-function relationships in GJCs. Modeling different connexin isoforms, simulating the transport process, and exploring molecular variants, have provided new hypotheses and out-of-the-box approaches to the study of these important channels.

Methods

Here, we review foundational structural studies and recent developments on GJCs using molecular modeling and simulation techniques, highlighting the methods and the cross-talk with experimental evidence.

Results and discussion

By comparing results obtained by molecular modeling and simulations techniques with structural and functional information obtained from both recent literature and structural databases, we provide a critical assesment of structure-function relationships that can be obtained from the junction between theoretical and experimental evidence.

     

Enzyme mimics complexing Cu(II) ion: structure-function relationships.

Five peptides containing (His-X2)-His or (His-X3)-His motifs have been designed and synthesized to coordinate Cu(II). Structural information was obtained by various spectroscopic techniques and was used as constraint to search for local conformational energy minima by molecular mechanics. Thermodynamic stability constants of the Cu(II) chelates was obtained by 19F-NMR. The synthesized Cu(II)-peptide chelates were tested as catalysts of some important red-ox processes occuring in biological systems, in particular oxidation of ascorbate and dismutation of superoxide ion. The catalytic efficiency of the five chelates was much lower than that of ascorbate oxidase. On the contrary, two of them showed kinetic constants for superoxide dismutation about one order of magnitude lower than that of the enzyme Cu,Zn superoxide dismutase. In both cases, the catalytic properties were dependent on the peptide sequence. The relationships between structure and activity are discussed to find the structural parameters crucial for catalytic activity that can be modulated by appropriate design and synthesis of the peptides.     

Immunity proteins to pore-forming colicins: structure-function relationships

Colicin A and B immunity proteins (Cai and Cbi, respectively) are homologous integral membrane proteins that interact within the core of the lipid bilayer with hydrophobic transmembrane helices of the corresponding colicin channel. By using various approaches (exchange of hydrophilic loops between Cai and Cbi, construction of Cbi/Cai hybrids, production of Cai as two fragments), we studied the structure-function relationships of Cai and Cbi. The results revealed unexpectedly high structural constraints for the function of these proteins. The periplasmic loops of Cai and Cbi did not carry the determinants for colicin recognition although most of these loops were required for Cai function; the cytoplasmic loop of Cai was found to be Involved in topology and function of Cai. The immunity function did not seem to be confined to a particular region of the immunity proteins.     

Influenza A virus M2 ion channel protein: a structure-function analysis.

A structure-function analysis of the influenza A virus M2 ion channel protein was performed. The M2 protein of human influenza virus A/Udorn/72 and mutants containing changes on one face of the putative alpha helix of the M2 transmembrane (TM) domain, several of which lead to amantadine resistance when found in virus, were expressed in oocytes of Xenopus laevis. The membrane currents of oocytes expressing mutant M2 ion channels were measured at both normal and low pH, and the amantadine-resistant mutant containing the change of alanine at residue 30 to threonine was found to have a significantly attenuated low pH activation response. The specific activity of the channel current of the amantadine-resistant mutants was investigated by measuring the membrane current of individual oocytes followed by quantification of the amount of M2 protein expressed in these single oocytes by immunoblotting analysis. The data indicate that changing residues on this face of the putative alpha helix of the M2 TM domain alters properties of the M2 ion channel. Some of the M2 proteins containing changes in the TM domain were found to be modified by addition of an N-linked carbohydrate chain at an asparagine residue that is membrane proximal and which is not modified in the wild-type M2 protein. These N-linked carbohydrate chains were further modified by addition of polylactosaminoglycan. A glycosylated M2 mutant protein (M2 + V, A30T) exhibited an ion channel activity with a voltage-activated, time-dependent kinetic component. Prevention of carbohydrate addition did not affect the altered channel activity. The ability of the M2 protein to tolerate deletions in the TM domain was examined by expressing three mutants (del29-31, del28-31, and del27-31) containing deletions of three, four, and five residues in the TM domain. No ion channel activity was detected from expression of M2 del29-31 and del27-31, whereas expression of M2 del28-31 resulted in an ion channel activity that was activated by hyperpolarization (and not low pH) and was resistant to amantadine block. Examination of the oligomeric form of M2 del28-31 indicated that the oligomer is different from wild-type M2, and the data were consistent with M2 del28-31 forming a pentamer.     

Establishing structure-function relationships for eumelanin

The aggregation-dependent optical properties of eumelanin from human hair are examined. When aggregation is increased, the absorption spectrum extends to lower energy. The absorption spectra of oligomers isolated from black human hair and Sepia officinalis are comparable and are in quantitative agreement with the reported action spectra for photoinduced oxygen consumption and free-radical generation by eumelanin. The agreement between the optical properties of human hair and squid eumelanins suggests the fundamental molecular constituents of the pigments are similar and aggregation-dependent photophysical behavior is a general feature of all eumelanins.     

Apolipoprotein A-I: structure-function relationships

The inverse relationship between high density lipoprotein (HDL) plasma levels and coronary heart disease has been attributed to the role that HDL and its major constituent, apolipoprotein A-I (apoA-I), play in reverse cholesterol transport (RCT). The efficiency of RCT depends on the specific ability of apoA-I to promote cellular cholesterol efflux, bind lipids, activate lecithin:cholesterol acyltransferase (LCAT), and form mature HDL that interact with specific receptors and lipid transfer proteins. From the intensive analysis of apoA-I secondary structure has emerged our current understanding of its different classes of amphipathic alpha-helices, which control lipid-binding specificity. The main challenge now is to define apoA-I tertiary structure in its lipid-free and lipid-bound forms. Two models are considered for discoidal lipoproteins formed by association of two apoA-I with phospholipids. In the first or picket fence model, each apoA-I wraps around the disc with antiparallel adjacent alpha-helices and with little intermolecular interactions. In the second or belt model, two antiparallel apoA-I are paired by their C-terminal alpha-helices, wrap around the lipoprotein, and are stabilized by multiple intermolecular interactions. While recent evidence supports the belt model, other models, including hybrid models, cannot be excluded. ApoA-I alpha-helices control lipid binding and association with varying levels of lipids. The N-terminal helix 44-65 and the C-terminal helix 210-241 are recognized as important for the initial association with lipids. In the central domain, helix 100-121 and, to a lesser extent, helix 122-143, are also very important for lipid binding and the formation of mature HDL, whereas helices between residues 144 and 186 contribute little. The LCAT activation domain has now been clearly assigned to helix 144-165 with secondary contribution by helix 166-186. The lower lipid binding affinity of the region 144-186 may be important to the activation mechanism allowing displacement of these apoA-I helices by LCAT and presentation of the lipid substrates. No specific sequence has been found that affects diffusional efflux to lipid-bound apoA-I. In contrast, the C-terminal helices, known to be important for lipid binding and maintenance of HDL in circulation, are also involved in the interaction of lipid-free apoA-I with macrophages and specific lipid efflux. While much progress has been made, other aspects of apoA-I structure-function relationships still need to be studied, particularly its lipoprotein topology and its interaction with other enzymes, lipid transfer proteins and receptors important for HDL metabolism.     

Interleukin-6: structure-function relationships.

Interleukin-6 (IL-6) is a multifunctional cytokine that plays a central role in host defense due to its wide range of immune and hematopoietic activities and its potent ability to induce the acute phase response. Overexpression of IL-6 has been implicated in the pathology of a number of diseases including multiple myeloma, rheumatoid arthritis, Castleman's disease, psoriasis, and post-menopausal osteoporosis. Hence, selective antagonists of IL-6 action may offer therapeutic benefits. IL-6 is a member of the family of cytokines that includes interleukin-11, leukemia inhibitory factor, oncostatin M, cardiotrophin-1, and ciliary neurotrophic factor. Like the other members of this family, IL-6 induces growth or differentiation via a receptor-system that involves a specific receptor and the use of a shared signaling subunit, gp130. Identification of the regions of IL-6 that are involved in the interactions with the IL-6 receptor, and gp130 is an important first step in the rational manipulation of the effects of this cytokine for therapeutic benefit. In this review, we focus on the sites on IL-6 which interact with its low-affinity specific receptor, the IL-6 receptor, and the high-affinity converter gp130. A tentative model for the IL-6 hexameric receptor ligand complex is presented and discussed with respect to the mechanism of action of the other members of the IL-6 family of cytokines.     

Protein tyrosine phosphatases: structure-function relationships

Structural analysis of protein tyrosine phosphatases (PTPs) has expanded considerably in the last several years, producing more than 200 structures in this class of enzymes (from 35 different proteins and their complexes with ligands). The small-medium size of the catalytic domain of approximately 280 residues plus a very compact fold makes it amenable to cloning and overexpression in bacterial systems thus facilitating crystallographic analysis. The low molecular weight PTPs being even smaller, approximately 150 residues, are also perfect targets for NMR analysis. The availability of different structures and complexes of PTPs with substrates and inhibitors has provided a wealth of information with profound effects in the way we understand their biological functions. Developments in mammalian expression technology recently led to the first crystal structure of a receptor-like PTP extracellular region. Altogether, the PTP structural work significantly advanced our knowledge regarding the architecture, regulation and substrate specificity of these enzymes. In this review, we compile the most prominent structural traits that characterize PTPs and their complexes with ligands. We discuss how the data can be used to design further functional experiments and as a basis for drug design given that many PTPs are now considered strategic therapeutic targets for human diseases such as diabetes and cancer.     

ATP synthase: structure-function relationships.

Recent work has focused on obtaining a better understanding of the three-dimensional structural relationships between the alpha and beta subunits of the F1 moiety and the location of nucleotide binding domains within these subunits. Four types of approach are currently being pursued: X-ray crystallographic, chemical, molecular biological and biochemical. Here we briefly review some of the major conclusions of these studies, and point out some of the problems that must be resolved before an adequate model that relates structure to function in the ATP synthase molecule can be formulated.     

Permeating disciplines: Overcoming barriers between molecular simulations and classical structure-function approaches in biological ion transport

Ion translocation across biological barriers is a fundamental requirement for life. In many cases, controlling this process—for example with neuroactive drugs—demands an understanding of rapid and reversible structural changes in membrane-embedded proteins, including ion channels and transporters. Classical approaches to electrophysiology and structural biology have provided valuable insights into several such proteins over macroscopic, often discontinuous scales of space and time. Integrating these observations into meaningful mechanistic models now relies increasingly on computational methods, particularly molecular dynamics simulations, while surfacing important challenges in data management and conceptual alignment. Here, we seek to provide contemporary context, concrete examples, and a look to the future for bridging disciplinary gaps in biological ion transport. This article is part of a Special Issue entitled: Beyond the Structure-Function Horizon of Membrane Proteins edited by Ute Hellmich, Rupak Doshi and Benjamin McIlwain.     

Glucagon structure-function relationships using isolated rat hepatocytes

The ability of glucagon and several of its semi-synthetic analogues to stimulate glucose production in isolated rat hepatocytes was measured and compared for relative potencies. The order of decreasing biological activities of glucagon in this assay was as follows: glucagon > [HArg¹²]-glucagon > [des-Asn²⁸, Thr²⁹][homoserinehydrazide²⁷]-glucagon approx. equal to [des-His¹]-glucagon > [des-Asn²⁸, Thr²⁹] [homoserinelactone²⁷]-glucagon > [des-Asn²⁸, Thr²⁹]-[n-butylhomoserineamide²⁷]-glucagon > glucagon_1?21. Qualititatively, these results are similar to those obtained previously in the hepatic plasma membrane adenylate cylase assay. Minor exceptions were noted for the hydrazide derivative nd the partial agonist [des-His¹]-glucagon, both of which were slightl y more potent relative to glucagon in the glycogenolytic assay than in the adenylate cyclase assay. The assay provides important insight into glucagon structure-function relationships.     

Glycomics: an integrated systems approach to structure-function relationships of glycans

In comparison with genomics and proteomics, the advancement of glycomics has faced unique challenges in the pursuit of developing analytical and biochemical tools and biological readouts to investigate glycan structure-function relationships. Glycans are more diverse in terms of chemical structure and information density than are DNA and proteins. This diversity arises from glycans' complex nontemplate-based biosynthesis, which involves several enzymes and isoforms of these enzymes. Consequently, glycans are expressed as an 'ensemble' of structures that mediate function. Moreover, unlike protein-protein interactions, which can be generally viewed as 'digital' in regulating function, glycan-protein interactions impinge on biological functions in a more 'analog' fashion that can in turn 'fine-tune' a biological response. This fine-tuning by glycans is achieved through the graded affinity, avidity and multivalency of their interactions. Given the importance of glycomics, this review focuses on areas of technologies and the importance of developing a bioinformatics platform to integrate the diverse datasets generated using the different technologies to allow a systems approach to glycan structure-function relationships.     

The CCN family of proteins: structure-function relationships

The CCN proteins are key signalling and regulatory molecules involved in many vital biological functions, including cell proliferation, angiogenesis, tumourigenesis and wound healing. How these proteins influence such a range of functions remains incompletely understood but is probably related to their discrete modular nature and a complex array of intra- and inter-molecular interactions with a variety of regulatory proteins and ligands. Although certain aspects of their biology can be attributed to the four individual modules that constitute the CCN proteins, recent results suggest that some of their biological functions require cooperation between modules. Indeed, the modular structure of CCN proteins provides important insight into their structure-function relationships.     

Protein kinase C pseudosubstrate prototope: structure-function relationships

A structure-function study of the protein kinase C (PK-C) pseudosubstrate sequence (R19FARK-GALRQKNV31) has been undertaken. The role of specific residues was investigated using an alanine substitution scan. Arg-22 was the most important determinant in the inhibitor sequence, since substitution of this residue by alanine gave a 600-fold increase in the IC50 value to 81 +/- 9 microM. Substitutions of other basic residue also increased the IC50, 5-, 11- and 24-fold for the Ala-19, Ala-23 and Ala-27 substitutions, respectively. The importance of basic residues in determining the potency of the pseudosubstrate peptide reflects the requirements for these residues in peptide substrate phosphorylation. The residues Gly-24, Leu-26 and Gln-28 were also important for pseudosubstrate inhibitor potency. The large difference in the IC50 value for the [A22]PK-C(19-31) peptide makes it a valuable control in studies employing the pseudosubstrate peptide to explore functional roles of PK-C.