Insights into the mechanism of C5aR inhibition by PMX53 via implicit solvent molecular dynamics simulations and docking.

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.

Structural mechanisms of constitutive activation in the C5a receptors with mutations in the extracellular loops: molecular modeling study.

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.

Simplified modeling approach suggests structural mechanisms for constitutive activation of the C5a receptor.

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.

Exploring new near-infrared fluorescent disulfide-based cyclic RGD peptide analogs for potential integrin-targeted optical imaging.

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(v)ß(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(v)ß(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(v)ß(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.

Constitutive activity of neural melanocortin receptors.

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.

Computational modeling of constitutively active mutants of GPCRs C5a receptor.

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.

Modeling the possible conformations of the extracellular loops in G-protein-coupled receptors.

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 beta-2 adrenergic receptor (beta2AR), turkey beta-1 adrenergic receptor (beta1AR), 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 conformers 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-molecular-weight ligands in beta2AR, beta1AR, 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.

Structural models for the complex of chemotaxis inhibitory protein of Staphylococcus aureus with the C5a receptor.

The study presents structural models for the complex of the chemotaxis inhibitory protein of Staphylococcus aureus, CHIPS, and receptor for anaphylotoxin C5a, C5aR. The models are based on the recently found NMR structure of the complex between CHIPS fragment 31-121 and C5aR fragment 7-28, as well as on previous results of molecular modeling of C5aR. Simple and straightforward modeling procedure selected low-energy conformations of the C5aR fragment 8-41 that simultaneously fit the NMR structure of the C5aR 10-18 fragment and properly orient the NMR structure of CHIPS(31-121) relative to C5aR. Extensive repacking of the side chains of CHIPS(31-121) and C5aR(8-41) predicted specific residue-residue interactions on the interface between CHIPS and C5aR. Many of these interactions were rationalized with experimental data obtained by site-directed mutagenesis of CHIPS and C5aR. The models correctly showed that CHIPS binds only to the first binding site of C5a to C5aR not competing with C5a fragment 59-74, which binds the second binding site of C5aR. The models also predict that two elements of CHIPS, fragments 48-58 and 97-111, may be used as structural templates for potential inhibitors of C5a.

Complement factor 5a receptor chimeras reveal the importance of lipid-facing residues in transport competence.

Residues that mediate helix-helix interactions within the seven transmembranes (TM) of G protein-coupled receptors are important for receptor biogenesis and the receptor switch mechanism. By contrast, the residues directly contacting the lipid bilayer have only recently garnered attention as potential receptor dimerization interfaces. In the present study, we aimed to determine the contributions of these lipid-facing residues to receptor function and oligomerization by systemically generating chimeric complement factor 5a receptors in which the entire lipid-exposed surface of a single TM helix was exchanged with the cognate residues from the angiotensin type 1 receptor. Disulfide-trapping and bioluminescence resonance energy transfer (BRET) studies demonstrated robust homodimerization of both complement factor 5a receptor and angiotensin type 1 receptor, but no evidence for heterodimerization. Despite relatively conservative substitutions, the lipid-facing chimeras (TM1, TM2, TM4, TM5, TM6 or TM7) were retained in the endoplasmic reticulum/cis-Golgi network. With the exception of the TM7 chimera that did not bind ligand, the lipid-facing chimeras bound ligand with low affinity, but similar to wild-type complement factor 5a receptors trapped in the endoplasmic reticulum with brefeldin A. These results suggest that the chimeric receptors were properly folded; moreover, native complement factor 5a receptors are not fully competent to bind ligand when present in the endoplasmic reticulum. BRET oligomerization studies demonstrated energy transfer between the wild-type complement factor 5a receptor and the lipid-facing chimeras, suggesting that the lipid-facing residues within a single TM segment are not essential for oligomerization. These studies highlight the importance of the lipid-facing residues in the complement factor 5a receptor for transport competence.

Modeling molecular mechanisms of binding of the anaphylatoxin C5a to the C5a receptor.

This study presents the 3D model of the complex between the anaphylatoxin C5a and its specific receptor, C5aR. This is the first 3D model of a G-protein-coupled receptor (GPCR) complex with a peptide ligand deduced by a molecular modeling procedure analyzing various conformational possibilities of the extracellular loops and the N-terminal segment of the GPCR. The modeling results indicated two very different ways of interacting between C5a and C5aR at the two interaction sites suggested earlier based on the data of site-directed mutagenesis. Specifically, C5a and C5aR can be involved in "mutual-induced fit", where the interface between the molecules is determined by both the receptor and the ligand. The rigid core of the C5a ligand selects the proper conformations of the highly flexible N-terminal segment of C5aR (the first interaction site). At the same time, the binding conformation of the flexible C-terminal fragment of C5a is selected by well-defined interactions with the TM region of the C5aR receptor (the second interaction site). The proposed 3D model of C5a/C5aR complex was built without direct use of structural constraints derived from site-directed mutagenesis reserving those data for validation of the model. The available data of site-directed mutagenesis of C5a and C5aR were successfully rationalized with the help of the model. Also, the modeling results predicted that the full-length C5a and C5a-des74 metabolite would have different binding modes with C5aR. Modeling approaches employed in this study are readily applicable for studies of molecular mechanisms of binding of other polypeptide ligands to their specific GPCRs.

Conformational movement of F251 contributes to the molecular mechanism of constitutive activation in the C5a receptor.

The activation mechanism of G-protein-coupled receptors triggered upon binding of a ligand represents a very important 'conformational switch' in the biological array of signal transduction. However, the molecular and functional details for this activation switch remain unknown. Random saturation mutagenesis data on the complement factor 5a receptor has provided a large data set of mutants including several constitutively active mutants. In the present study, we employed computational modeling to rationalize the constitutive activity for two constitutively active mutants, NQ (I124N/L127Q) and F251A, and we then made predictions for a series of mutants that either promote or constrain constitutive activity. Biological testing of the site-directed mutants confirmed most of the predictions of the computational modeling. These results support a molecular mechanism of constitutive activity in complement factor 5a receptor mutants that is associated with conformational changes in a network of residues neighboring F251 as the focal point of origin.

Structure of the complement factor 5a receptor-ligand complex studied by disulfide trapping and molecular modeling.

Complement factor 5a (C5a) is an anaphylatoxin that acts by binding to a G protein-coupled receptor, the C5aR. The relative orientation of this ligand-receptor pair is investigated here using the novel technique of disulfide trapping by random mutagenesis (DTRM) and molecular modeling. In the DTRM technique, an unpaired cysteine is introduced in the ligand, and a library of randomly mutagenized receptors is screened to identify mutants that introduce a cysteine at a position in the receptor that allows functional interactions with the ligand. By repeating this analysis at six positions of C5a, we identify six unique sets of intermolecular interactions for the C5a-C5aR complex, which are then compared with an independently developed computational three-dimensional model of the complex. This analysis reveals that the interface of the receptor N terminus with the cysteine-containing ligand molecules is selected from a variety of possible receptor conformations that exist in dynamic equilibrium. In contrast, DTRM identifies a single position in the second extracellular loop of the receptor that interacts specifically with a cysteine probe placed in the C-terminal tail of the C5a ligand.

Conformational templates for rational drug design: flexibility of cyclo(D-Pro1-Ala2-Ala3-Ala4-Ala5) in DMSO solution.

Long MD simulations (100 ns) for the important model cyclopentapeptide cyclo(D-Pro1-Ala2-Ala3-Ala4-Ala5) were performed in explicit DMSO solution using both OPLS-AA and AMBER03 force fields. Simulations revealed conformational transitions between two main conformers, a predominant one (population 93-99%) and a minor conformer (population 0.4-6.7%). These results are in excellent agreement with 20 experimental proton-proton distances estimated for this cyclopentapeptide. The previously discussed gamma-turn-like conformation for Ala4 was present only in a minor conformer.

Molecular modeling suggests conformational scaffolds specifically targeting five subtypes of somatostatin receptors.

Several analogs of somatostatin with conformational constraints in their peptide backbones have been modeled to determine energetically feasible conformations. Comparison of low-energy backbone structures of these peptides suggested unique conformations of the central Phe/Ala(i)-D-Trp(i+1)-Lys(i+2)-Thr(i+3) fragment characteristic for specific interactions of somatostatin with each of the five distinct subtypes of somatostatin receptors (SSTRs). The conformations obtained were in good agreement with experimental data obtained earlier by NMR measurements and/or X-ray crystallography. The results help rationalize experimental observations on the specificity of binding of various somatostatin analogs with different subtypes of the SSTRs. They also serve as templates for the design of conformationally constrained non-peptide scaffolds that effectively and selectively interact with different subtypes of SSTRs. Such scaffolds can be convenient carriers of radiolabels and near-infrared labels in specific agents for imaging tumors expressing different SSTR subtypes.

Modeling of the complex between transducin and photoactivated rhodopsin, a prototypical G-protein-coupled receptor.

Obtaining a reliable 3D model for the complex formed by photoactivated rhodopsin (R*) and its G-protein, transducin (Gtalphabetagamma), would significantly benefit the entire field of structural biology of G-protein-coupled receptors (GPCRs). In this study, we have performed extensive configurational sampling for the isolated C-terminal fragment of the alpha-subunit of transducin, Gtalpha 340-350, within cavities of photoactivated rhodopsin formed by different energetically feasible conformations of the intracellular loops. Our results suggested a new 3D model of the rhodopsin-transducin complex that fully satisfied all available experimental data on site-directed mutagenesis of rhodopsin and Gtalphabetagamma as well as data from disulfide-linking experiments. Importantly, the experimental data were not used as a priori constraints in model building. We performed a thorough comparison of existing computational models of the rhodopsin-transducin complex with each other and with current experimental data. It was found that different models suggest interactions with different molecules in the rhodopsin oligomer, that providing valuable guidance in design of specific novel experimental studies of the R*-Gtalphabetagamma complex. Finally, we demonstrated that the isolated Gtalpha 340-350 fragment does not necessarily bind rhodopsin in the same binding mode as the same segment in intact Gtalpha.

Defining the interface between the C-terminal fragment of alpha-transducin and photoactivated rhodopsin.

A novel combination of experimental data and extensive computational modeling was used to explore probable protein-protein interactions between photoactivated rhodopsin (R*) and experimentally determined R*-bound structures of the C-terminal fragment of alpha-transducin (Gt(alpha)(340-350)) and its analogs. Rather than using one set of loop structures derived from the dark-adapted rhodopsin state, R* was modeled in this study using various energetically feasible sets of intracellular loop (IC loop) conformations proposed previously in another study. The R*-bound conformation of Gt(alpha)(340-350) and several analogs were modeled using experimental transferred nuclear Overhauser effect data derived upon binding R*. Gt(alpha)(340-350) and its analogs were docked to various conformations of the intracellular loops, followed by optimization of side-chain spatial positions in both R* and Gt(alpha)(340-350) to obtain low-energy complexes. Finally, the structures of each complex were subjected to energy minimization using the OPLS/GBSA force field. The resulting residue-residue contacts at the interface between R* and Gt(alpha)(340-350) were validated by comparison with available experimental data, primarily from mutational studies. Computational modeling performed for Gt(alpha)(340-350) and its analogs when bound to R* revealed a consensus of general residue-residue interactions, necessary for efficient complex formation between R* and its Gt(alpha) recognition motif.

A comprehensive structure-function map of the intracellular surface of the human C5a receptor. II. Elucidation of G protein specificity determinants.

Within any given cell many G protein-coupled receptors are expressed in the presence of multiple G proteins, yet most receptors couple to a specific subset of G proteins to elicit their programmed response. Numerous studies demonstrate that the carboxyl-terminal five amino acids of the Galpha subunits are a major determinant of specificity, however the receptor determinants of specificity are less clear. We have used a collection of 133 functional mutants of the C5a receptor obtained in a mutagenesis screen targeting the intracellular loops and the carboxyl terminus (Matsumoto, M. L., Narzinski, K., Kiser, P. D., Nikiforovich, G. V., and Baranski, T. J. (2007) J. Biol. Chem. 282, 3105-3121) to investigate how specificity is encoded. Each mutant, originally selected for its ability to signal through a nearly full-length Galpha(i) in yeast, was tested to see whether it could activate three versions of chimeric Galpha subunits consisting of Gpa1 fused to the carboxyl-terminal five amino acids of Galpha(i), Galpha(q), or Galpha(s) in yeast. Surprisingly the carboxyl-terminal tail of the C5a receptor is the most important specificity determinant in that nearly all mutants in this region showed a gain in coupling to Galpha(q) and/or Galpha(s). More than half of the receptors mutated in the second intracellular loop also demonstrated broadened G protein coupling. Given a lack of selective advantage for this broadened signaling in the initial screen, we propose a model in which the carboxyl-terminal tail acts together with the intracellular loops to generate a specificity filter for receptor-G protein interactions that functions primarily to restrict access of incorrect G proteins to the receptor.

A comprehensive structure-function map of the intracellular surface of the human C5a receptor. I. Identification of critical residues.

G protein-coupled receptors are one of the largest protein families in nature; however, the mechanisms by which they activate G proteins are still poorly understood. To identify residues on the intracellular face of the human C5a receptor that are involved in G protein activation, we performed a genetic analysis of each of the three intracellular loops and the carboxyl-terminal tail of the receptor. Amino acid substitutions were randomly incorporated into each loop, and functional receptors were identified in yeast. The third intracellular loop contains the largest number of preserved residues (positions resistant to amino acid substitutions), followed by the second loop, the first loop, and lastly the carboxyl terminus. Surprisingly, complete removal of the carboxyl-terminal tail did not impair C5a receptor signaling. When mapped onto a three-dimensional structural model of the inactive state of the C5a receptor, the preserved residues reside on one half of the intracellular surface of the receptor, creating a potential activation face. Together these data provide one of the most comprehensive functional maps of the intracellular surface of any G protein-coupled receptor to date.

Interactions between conserved residues in transmembrane helices 2 and 7 during angiotensin AT1 receptor activation.

Site-directed mutagenesis studies and independent molecular modeling studies were combined to investigate the network of inter-residue interactions within the transmembrane region of the angiotensin AT(1a) receptor. Site-directed mutagenesis was focused on residues Tyr292, Asn294, Asn295, and Asn298 in transmembrane helix 7, and the conserved Asp74 in helix 2 and other polar residues. Functional interactions between pairs of residues were evaluated by determining the effects of single and double-reciprocal mutations on agonist-induced AT(1a) receptor activation. Replacement of Tyr292 by aspartate in helix 7 abolished radioligand binding to both Y292D and D74Y/Y292D mutant receptors. Reciprocal mutations of Asp74/Asn294, Ser115/Asn294, Ser252/Asn294, and Asn298/Sen115 caused additive impairment of function, suggesting that these pairs of residues make independent contributions to AT(1a) receptor activation. In contrast, mutations of the Asp74/Tyr298 pair revealed that the D74N/N298D reciprocal mutation substantially increased the impaired inositol phosphate responses of the D74N and N298D receptors. Extensive molecular modeling yielded 3D models of the TM region of the AT(1) receptor and the mutants as well as of their complexes with angiotensin II, which were used to rationalize the possible reasons of impairing of function of some mutants. These data indicate that Asp74 and Asn298 are not optimally positioned for direct strong interaction in the resting conformation of the AT(1a) receptor. Balance of interactions between residues in helix 2 (as D74) and helix 7 (as N294, N295 and N298) in the AT(1) receptors, however, has a crucial role both in determining their functional activity and levels of their expression.