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St. Louis, MO, United States

Nikiforovich G.V.,MolLife Design LLC | Baranski T.J.,University of Washington
Proteins: Structure, Function and Bioinformatics | Year: 2012

Previously we demonstrated by random saturation mutagenesis a set of mutations in the extracellular (EC) loops that constitutively activate the C5a receptor (C5aR) (Klco et al., Nat Struct Mol Biol 2005;12:320-326; Klco et al., J Biol Chem 2006;281:12010-12019). In this study, molecular modeling revealed possible conformations for the extracellular loops of the C5a receptors with mutations in the EC2 loop or in the EC3 loop. Comparison of low-energy conformations of the EC loops defined two distinct clusters of conformations typical either for strongly constitutively active mutants of C5aR (the CAM cluster) or for nonconstitutively active mutants (the non-CAM cluster). In the CAM cluster, the EC3 loop was turned towards the transmembrane (TM) helical bundle and more closely interacted with EC2 than in the non-CAM cluster. This suggested a structural mechanism of constitutive activity where EC3 contacts EC2 leading to EC2 interactions with helix TM3, thus triggering movement of TM7 towards TM2 and TM3. The movement initiates rearrangement of the system of hydrogen bonds between TM2, TM3 and TM7 including formation of the hydrogen bond between the side chains of D82 2.50 in TM2 and N296 7.49 in TM7, which is crucial for formation of the activated states of the C5a receptors (Nikiforovich et al., Proteins: Struct Funct Gene 2011;79:787-802). Since the relative large length of EC3 in C5aR (13 residues) is comparable with those in many other members of rhodopsin family of GPCRs (13-19 residues), our findings might reflect general mechanisms of receptor constitutive activation. The very recent X-ray structure of the agonist-induced constitutively active mutant of rhodopsin (Standfuss et al., Nature 2011;471:656-660) is discussed in view of our modeling results. © 2011 Wiley Periodicals, Inc. Source


Tamamis P.,University of Cyprus | Kieslich C.A.,University of California at Riverside | Nikiforovich G.V.,MolLife Design LLC | Woodruff T.M.,University of Queensland | And 2 more authors.
BMC Biophysics | Year: 2014

Background: The complement protein C5a acts by primarily binding and activating the G-protein coupled C5a receptor C5aR (CD88), and is implicated in many inflammatory diseases. The cyclic hexapeptide PMX53 (sequence Ace-Phe-[Orn-Pro-dCha-Trp-Arg]) is a full C5aR antagonist of nanomolar potency, and is widely used to study C5aR function in disease. Results: We construct for the first time molecular models for the C5aR:PMX53 complex without the a priori use of experimental constraints, via a computational framework of molecular dynamics (MD) simulations, docking, conformational clustering and free energy filtering. The models agree with experimental data, and are used to propose important intermolecular interactions contributing to binding, and to develop a hypothesis for the mechanism of PMX53 antagonism. Conclusion: This work forms the basis for the design of improved C5aR antagonists, as well as for atomic-detail mechanistic studies of complement activation and function. Our computational framework can be widely used to develop GPCR-ligand structural models in membrane environments, peptidomimetics and other chemical compounds with potential clinical use. © 2014 Tamamis et al.; licensee BioMed Central Ltd. Source


Ye Y.,University of Washington | Xu B.,University of Washington | Nikiforovich G.V.,University of Washington | Nikiforovich G.V.,MolLife Design LLC | And 2 more authors.
Bioorganic and Medicinal Chemistry Letters | Year: 2011

We synthesized disulfide-based cyclic RGD pentapeptides bearing a near-infrared fluorescent dye (cypate), represented by cypate-c(CRGDC) (1) for integrin-targeted optical imaging. These compounds were compared with the traditional lactam-based cyclic RGD counterpart, cypate-c(RGDfK) (2). Molecular modeling suggests that the binding affinity of 2 to integrin α vβ 3 is an order of magnitude higher than that of 1. This was confirmed experimentally, which further showed that substitution of Gly with Pro, Val and Tyr in 1 remarkably hampered the α vβ 3 binding. Interestingly, cell microscopy with A549 cells showed that 1 exhibited higher cellular staining than 2. These results indicate that factors other than receptor binding affinity to α vβ 3 dimeric proteins mediate cellular uptake. Consequently, 1 and its analogs may serve as valuable molecular probes for investigating the selectivity and specificity of integrin targeting by optical imaging. © 2011 Elsevier Ltd. All rights reserved. Source


Tao Y.-X.,Auburn University | Huang H.,Auburn University | Wang Z.-Q.,Auburn University | Wang Z.-Q.,Yangzhou University | And 3 more authors.
Methods in Enzymology | Year: 2010

The two neural melanocortin receptors (MCRs), melanocortin-3 and -4 receptors (MC3R and MC4R), are G protein-coupled receptors expressed primarily in the brain that regulate different aspects of energy homeostasis. The MCRs are unique in having endogenous antagonists, agouti and agouti-related protein (AgRP). These antagonists were later shown to be inverse agonists. The MC3R has little or no constitutive activity, whereas the MC4R has significant constitutive activity that can easily be detected. We describe herein methods for detecting constitutive activities in these receptors and small molecule ligands as inverse agonists. AgRP is an inverse agonist for both MC3R and MC4R. We also provide models for the constitutively active MC4R mutants. © 2010 Elsevier Inc. Source


Nikiforovich G.V.,MolLife Design LLC | Marshall G.R.,University of Washington | Baranski T.J.,University of Washington
Proteins: Structure, Function and Bioinformatics | Year: 2011

Molecular modeling of conformational changes occurring in the transmembrane region of the complement factor 5a receptor (C5aR) during receptor activation was performed by comparing two constitutively active mutants (CAMs) of C5aR, NQ (I124N/L127Q), and F251A, to those of the wild-type C5aR and NQ-N296A (I124N/L127Q/N296A), which have the wild-type phenotype. Modeling involved comprehensive sampling of various rotations of TM helices aligned to the crystal template of the dark-adapted rhodopsin along their long axes. By assuming that the relative energies of the spontaneously activated states of CAMs should be lower or at least comparable to energies characteristic for the ground states, we selected the plausible models for the conformational states associated with constitutive activation in C5aR. The modeling revealed that the hydrogen bonds between the side chains of D82-N119, S85-N119, and S131-C221 characteristic for the ground state were replaced by the hydrogen bonds D82-N296, N296-Y300, and S131-R134, respectively, in the activated states. Also, conformational transitions that occurred upon activation were hindered by contacts between the side chains of L127 and F251. The results rationalize the available data of mutagenesis in C5aR and offer the first specific molecular mechanism for the loss of constitutive activity in NQ-N296A. Our results also contributed to understanding the general structural mechanisms of activation in G-protein-coupled receptors lacking the "ionic lock", R 3.50 and E/D 6.30. Importantly, these results were obtained by modeling approaches that deliberately simplify many elements in order to explore potential conformations of GPCRs involving large-scale molecular movements. © 2010 Wiley-Liss, Inc. Source

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