MolLife Design LLC

St. Louis, MO, United States

MolLife Design LLC

St. Louis, MO, United States
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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.


PubMed | University of California at Riverside, University of Queensland, University of Cyprus and MolLife Design LLC
Type: | Journal: BMC biophysics | Year: 2014

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.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.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.


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.


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.


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.


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.


Nikiforovich G.V.,MolLife Design LLC | Taylor C.M.,University of Washington | Marshall G.R.,University of Washington | Baranski T.J.,University of Washington
Proteins: Structure, Function and Bioinformatics | Year: 2010

This study presents the results of a de novo approach modeling possible conformational dynamics of the extracellular (EC) loops in G-protein-coupled receptors (GPCRs), specifically in bovine rhodopsin (bRh), squid rhodopsin (sRh), human β-2 adrenergic receptor (β2AR), turkey β-1 adrenergic receptor (β1AR), and human A2 adenosine receptor (A2AR). The approach deliberately sacrificed a detailed description of any particular 3D structure of the loops in GPCRs in favor of a less precise description of many possible structures. Despite this, the approach found ensembles of the low-energy conformen of the EC loops that contained structures close to the available X-ray snapshots. For the smaller EC1 and EC3 loops (6-11 residues), our results were comparable with the best recent results obtained by other authors using much more sophisticated techniques. For the larger EC2 loops (25-34 residues), our results consistently yielded structures significantly closer to the X-ray snapshots than the results of the other authors for loops of similar size. The results suggested possible large-scale movements of the EC loops in GPCRs that might determine their conformational dynamics. The approach was also validated by accurately reproducing the docking poses for low-molecularweight ligands in β2AR, βlAR, and A2AR, demonstrating the possible influence of the conformations of the EC loops on the binding sites of ligands. The approach correctly predicted the system of disulfide bridges between the EC loops in A2AR and elucidated the probable pathways for forming this system. © 2009 Wiley-Liss, Inc.


Nikiforovich G.V.,MolLife Design LLC | Baranski T.J.,University of Washington
Methods in Enzymology | Year: 2010

In the past decade, an increasing number of studies using computational modeling procedures have focused on the structural aspects of constitutive activity in G protein-coupled receptors (GPCRs). This chapter reviews various conceptual approaches in computational modeling of constitutively active mutants (CAMs) including analyzing three-dimensional models of the ground states of GPCRs based on structural homology with the known X-ray templates; molecular dynamics simulations starting from the ground states; and modeling of CAMs based on the experimentally suggested templates of the possible activated states. The developed buildup procedure of rotational sampling of the TM regions of GPCRs is highlighted in more detail. Experimental data on CAMs of the complement factor 5a receptor (C5aR) are used to validate the rotational sampling results. © 2010 Elsevier Inc.


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.


PubMed | MolLife Design LLC
Type: Journal Article | Journal: Proteins | 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.

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