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G-protein coupled receptor Dynamics

"GPCR are not simple on/off switches but highly dynamic structures that exist in equilibriums between active and inactive conformations" Gether, 2000


Characterization of complexity of conformational states and their dynamics by single molecule spectroscopy:

Peleg, G., P. Ghanouni, B.K. Kobilka, and R.N. Zare, Single-molecule spectroscopy of the beta(2) adrenergic receptor: observation of conformational substates in a membrane protein. Proc Natl Acad Sci U S A, 2001. 98(15): p. 8469-74

- use of single moelcule spectroscopy: overcome the problem of ensemble averaging of other methods, ref. 

1.         Nie, S. and R.N. Zare, Optical detection of single molecules. Annu Rev Biophys Biomol Struct, 1997. 26: p. 567-96.

2.         Nie, S., D.T. Chiu, and R.N. Zare, Probing individual molecules with confocal fluorescence microscopy. Science, 1994. 266(5187): p. 1018-21.

3.            Dickson, R.M., A.B. Cubitt, R.Y. Tsien, and W.E. Moerner, On/off blinking and switching behaviour of single molecules of green fluorescent protein. Nature, 1997. 388(6640): p. 355-8.

4.         Xie, X.S. and J.K. Trautman, Annu Rev Phys Chem, 1998. 49: p. 441-480.

- beta2-adrenergic receptor

- fluorescence tag in detergent micelles at position Cys265 (Fluorescein-5-maleimide) at the end of TM VI

- of 13 cysteines, Cys265 is the only accessible to the Fluorescein-5-maleimide (5 in TM region, 4 in disulfide bonds in EC domain, 1 in C-term is palmitoylated, remaining 2 C-terminal Cys form a disulfide bond during purification)

- at least two distinct substates for the native receptor,  "is a conformationally flexible molecule", 2 predominant, several minor ones possibly represented by different burst intensities

- full agonist ISO (which has higher binding affinity KI~10 microM and higher biological efficacy than adrenaline) stabilizes confrmational substates that are rare in the native receptor

- conformational changes associated with agonist binding result in a marked change in the distribution of photon-burst sizes

- conformational heterogeneity of GPCR in the presence and absence of a bound agonist

- various ligands (agonists and antagonists) change both the shape of the entire distribution and the populations of the conformational substaes



Conclusions on Dynamics from Disulfide bond crosslinking

1.     disulfide crosslinking in muscarinic receptor ACM2 and ACM3 (Zeng, Hopp et al. 1999) also see GPCRreviews.html

    method: visualization of receptor by antibody tag in cell lysates

    added Factor Xa cleavage site in third CP loop, just before the second cysteines, so that disulfide crosslinked receptor migrates at same position as native, while non-crosslinked migrates at fragment positions

    analogous to the Farrens study - one cysteine at CP end of helix 3, and set of second cysteine position at CP end of helix 6

    8 out of 10 mutants formed disulfide bond with copper-phenantroline

    functional consequence: blocks activation drastically where disulfides formed (same as in rhodopsin)

    notion of dynamics in CP face that allows disulfide bonds to form


Conformational heterogeneity in Beta2-adrenergic receptor and functional consequences: Ghanouni, P., Z. Gryczynski, et al. (2001). "Functionally different agonists induce distinct conformations in the G protein coupling domain of the beta 2 adrenergic receptor." J Biol Chem 276(27): 24433-6.

- fluorescent labeled beta2-adrenergic receptor in CP domain (EF-loop: Cys265)

- in absence of ligands: oscillation around a single detectable conformation: this may explain difficulty in obtaining crystals in the absence of a bound ligand

- binding of antagonist: no change in the conformation, but reduction it the flexibility of the domain: this may represent the energetically more stable conformation and may not be the maximally active conformation

- binding of full agonist: two distinct conformations

- binding of partial agonists: different conformations than when full agonist binds

=> GPCR are plastic: there may be a large spectrum of possible conformations that could be stabilized by drugs

- also see GPCR activation


Speculation on role of dynamics for GPCR activation (also see GPCR activation):

Gether, U. (2000). "Uncovering molecular mechanisms involved in activation of G protein-coupled receptors." Endocr Rev 21(1): 90-113.


Dynamics in Dopamine D2 receptor (see review by Gether, 2000), reference: Javitch, J.A., D. Fu, and J. Chen, Residues in the fifth membrane-spanning segment of the dopamine D2 receptor exposed in the binding-site crevice. Biochemistry, 1995. 34(50): p. 16433-9.

using MTS accessibility, the pattern in TM V was different from that in the other helices,, stretch of 10 consecutive aa in the outer portion of TM V was exposed in the binding crevice, which is not as predicted (one side should be exposed and the other side hidden from the crevice)

it was proposed that TM V may be structurally flexible and rapidly shift between different conformations, exposing different sets of residues to the binding crevice

the exposed region contains residues believed to form key contacts with the small molecule agonists


Interconversion of different conformations of receptors

Class A peptide receptors, e.g. NK-1 receptor:

    - mutations can affect the ability of the receptor to feely interchange between distinct receptor conformations, which bind the non-peptide antagonist and peptide agonist with high affinity, respectively, ref.

        Rosenkilde, M.M., M. Cahir, U. Gether, S.A. Hjorth, and T.W. Schwartz, Mutations along transmembrane segment II of the NK-1 receptor affect substance P competition with non-peptide antagonists but not substance P binding. J Biol Chem, 1994. 269(45): p. 28160-4

    - clearly different receptor conformational states that display distinct selectivity for the tachykinin peptides (NK-1 receptor): ref. 

            Ciucci, A., C. Palma, S. Manzini, and T.M. Werge, Point mutation increases a form of the NK1 receptor with high affinity for neurokinin A and B and septide. Br J Pharmacol, 1998. 125(2): p. 393-401.

    Ciucci, A., C. Palma, D. Riitano, S. Manzini, and T.M. Werge, Gly166 in the NK1 receptor regulates tachykinin selectivity and receptor conformation. FEBS Lett, 1997. 416(3): p. 335-8

    - similar observations in kappa-opioid receptor , ref.

        Hjorth, S.A., K. Thirstrup, and T.W. Schwartz, Radioligand-dependent discrepancy in agonist affinities enhanced by mutations in the kappa-opioid receptor. Mol Pharmacol, 1996. 50(4): p. 977-84


Constraining mutations (see review by Gether, 2000):

- molecular mechanism of constitutive activity: Ala293 VI.0/6.34 in C-terminal part of C-III of alpha1B-adrenergic receptor was substituted by all 20 aa, and all have higher basal activity (Lefkowitz et al: Kjelsberg, M.A., S. Cotecchia, J. Ostrowski, M.G. Caron, and R.J. Lefkowitz, Constitutive activation of the alpha 1B-adrenergic receptor by all amino acid substitutions at a single site. Evidence for a region which constrains receptor activation. J Biol Chem, 1992. 267(3): p. 1430-3.) ==> constraining intramolecular interaction have been conserved to keep receptor in an inactive conformation in the absence of agonist and these inactivating constraints could be released as  a part of the receptor activation mechanism, either after agonist binding of due to specific mutations, causing key sequences to be exposed to the G protein

- hypothesis supported by dynamics: 

constitutively activated beta2-adrenergic receptor mutation are characterized by a marked structural instability and enhanced conformational flexibility of the purified receptor proteins, refs.: 

Rasmussen, S.G., A.D. Jensen, G. Liapakis, P. Ghanouni, J.A. Javitch, and U. Gether, Mutation of a highly conserved aspartic acid in the beta2 adrenergic receptor: constitutive activation, structural instability, and conformational rearrangement of transmembrane segment 6. Mol Pharmacol, 1999. 56(1): p. 175-84

Gether, U., J.A. Ballesteros, R. Seifert, E. Sanders-Bush, H. Weinstein, and B.K. Kobilka, Structural instability of a constitutively active G protein-coupled receptor. Agonist-independent activation due to conformational flexibility. J Biol Chem, 1997. 272(5): p. 2587-90

==> the mutations have disrupted intramolecular interactions allowing the receptor to undergo conversion more readily between the inactive and active state


2-state model not sufficient to explain complex behavior of GPCR, but rather there are multiple conformational states of GPCR (Review in Gether, 2000 - References:)

1.         Scheer, A., F. Fanelli, T. Costa, P.G. De Benedetti, and S. Cotecchia, Constitutively active mutants of the alpha 1B-adrenergic receptor: role of highly conserved polar amino acids in receptor activation. Embo J, 1996. 15(14): p. 3566-78.

2.         Gether, U., J.A. Lowe, 3rd, and T.W. Schwartz, Tachykinin non-peptide antagonists: binding domain and molecular mode of action. Biochem Soc Trans, 1995. 23(1): p. 96-102.

3.            Chidiac, P., T.E. Hebert, M. Valiquette, M. Dennis, and M. Bouvier, Inverse agonist activity of beta-adrenergic antagonists. Mol Pharmacol, 1994. 45(3): p. 490-9.

4.            Riitano, D., T.M. Werge, and T. Costa, A mutation changes ligand selectivity and transmembrane signaling preference of the neurokinin-1 receptor. J Biol Chem, 1997. 272(12): p. 7646-55.

5.         Reale, V., F. Hannan, L.M. Hall, and P.D. Evans, Agonist-specific coupling of a cloned Drosophila melanogaster D1-like dopamine receptor to multiple second messenger pathways by synthetic agonists. J Neurosci, 1997. 17(17): p. 6545-53.

6.         Wiens, B.L., C.S. Nelson, and K.A. Neve, Contribution of serine residues to constitutive and agonist-induced signaling via the D2S dopamine receptor: evidence for multiple, agonist-specific active conformations. Mol Pharmacol, 1998. 54(2): p. 435-44.

7.            Mhaouty-Kodja, S., L.S. Barak, A. Scheer, L. Abuin, D. Diviani, M.G. Caron, and S. Cotecchia, Constitutively active alpha-1b adrenergic receptor mutants display different phosphorylation and internalization features. Mol Pharmacol, 1999. 55(2): p. 339-47.

- different constitutively active mutants of the alpha1B-receptor are differentially phosphorylated and internalized although they convey a similar agonist-independent activity

- direct structural evidence from fluorescence spectroscopy of purified beta2-adrenergic receptor, ref. Gether, U., J.A. Lowe, 3rd, and T.W. Schwartz, Tachykinin non-peptide antagonists: binding domain and molecular mode of action. Biochem Soc Trans, 1995. 23(1): p. 96-102.



General Comments on Dynamics:

Proteins that are functionally in a single conformation state undergo small conformational fluctuations around a minimum energy state: Frauenfelder, H., S. G. Sligar, et al. (1991). "The energy landscapes and motions of proteins." Science 254(5038): 1598-603. 

conformational fluctuations may be due to local unfolding within 3d structure: Freire, E. (2000). "Can allosteric regulation be predicted from structure?" Proc Natl Acad Sci U S A 97: 11680-11683.

- experimental evidence: D/H exchange by NMR, fluorescence lifetime measurements (See beta2-adrenergic receptor above and under GPCR activation)

- view pioneered by Englander:

    - proteins undergo local unfolding scattered throughout their entire structure

    - the unfolding reactions occur independently of each other

    - may involve only a few residues (3-5 amino acids) or entire protein

=> native state ensemble

- consequence: Gibbs energy of stabilization of a protein is not uniformly distributed, there re regions with high and with low stability constants

- under native conditions, cooperativity appear to be local rather than global

- COREX algorithm 

    - considers the native state as a statistical ensemble in which each state is characterized by having some region(s) unfolded

    - produces a list of possible conformation and their probability distribution function

    - can be used to study the effect of ligands on that distribution

- COREX gives the distribution if a single protein molecule were observed over a period sufficiently long for thermodynamic averaging

- COREX therefore allows ensemble properties to be mapped into individual molecules

- CORE_BIND algorithm

    - predicts the propagation of binding effects though the structure upon binding (e.g. lysozyme-antibody complex)

    - ligands selectively bind to those states that possess the selected functional property (inactive or active etc.) and thereby cause a change in the probability distribution of states

- Long-range communication

    - communication between sites accomplished by correlation between binding competencies

    - example: in glycerol kinase, an allosteric inhibitor binds at a site more than 30 A away from active site

    - COREX and CORE_BIND show that only a small set of residues are involved in the allosteric coupling between regulatory and catalytic sites

=> long-range communication in proteins can be transmitted via few amino acids