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1.  Derivation of ligands for the complement C3a receptor from the C-terminus of C5a 
The complement cascade is a highly sophisticated network of proteins that are well regulated and directed in response to invading pathogens or tissue injury. Complement C3a and C5a are key mediators produced by this cascade, and their dysregulation has been linked to a plethora of inflammatory and autoimmune diseases. Consequently, this has stimulated interest in the development of ligands for the receptors for these complement peptides, C3a receptor, and C5a1 (C5aR/CD88). In this study we used computational methods to design novel C5a1 receptor ligands. However, functional screening in human monocyte-derived macrophages using the xCELLigence label-free platform demonstrated altered specificity of our ligands. No agonist/antagonist activity was observed at C5a1, but we instead saw that the ligands were able to partially agonize the closely related complement receptor C3a receptor. This was verified in the presence of C3a receptor antagonist SB 290157 and in a stable cell line expressing either C5a1 or C3a receptor alone. C3a agonism has been suggested to be a potential treatment of acute neutrophil-driven traumatic pathologies, and may have great potential as a therapeutic avenue in this arena.
doi:10.1016/j.ejphar.2014.10.041
PMCID: PMC4263610  PMID: 25446428
C3a receptor; C5a1 receptor; Peptide design; In silico sequence selection; Computational optimization; Label-free screening
2.  Novel Compstatin Family Peptides Inhibit Complement Activation by Drusen-Like Deposits in Human Retinal Pigmented Epithelial Cell Cultures 
Experimental eye research  2013;0:10.1016/j.exer.2013.07.023.
We have used a novel human retinal pigmented epithelial (RPE) cell-based model that mimics drusen biogenesis and the pathobiology of age-related macular degeneration to evaluate the efficacy of newly designed peptide inhibitors of the complement system. The peptides belong to the compstatin family and, compared to existing compstatin analogs, have been optimized to promote binding to their target, complement protein C3, and to enhance solubility by improving their polarity/hydrophobicity ratios. Based on analysis of molecular dynamics simulation data of peptide-C3 complexes, novel binding features were designed by introducing intermolecular salt bridge-forming arginines at the N-terminus and at position -1 of N-terminal dipeptide extensions. Our study demonstrates that the RPE cell assay has discriminatory capability for measuring the efficacy and potency of inhibitory peptides in a macular disease environment.
doi:10.1016/j.exer.2013.07.023
PMCID: PMC3840162  PMID: 23954241
macular degeneration; drusen; retinal pigmented epithelium; complement system; complement inhibitors; compstatin family peptides; molecular dynamics
3.  Electrostatic Modeling Predicts the Activities of Orthopoxvirus Complement Control Proteins1 
Regulation of complement activation by pathogens and the host are critical for survival. Using two highly related orthopoxvirus proteins, the vaccinia and variola (smallpox) virus complement control proteins, which differ by only 11 aa, but differ 1000-fold in their ability to regulate complement activation, we investigated the role of electrostatic potential in predicting functional activity. Electrostatic modeling of the two proteins predicted that altering the vaccinia virus protein to contain the amino acids present in the second short consensus repeat domain of the smallpox protein would result in a vaccinia virus protein with increased complement regulatory activity. Mutagenesis of the vaccinia virus protein confirmed that changing the electrostatic potential of specific regions of the molecule influences its activity and identifies critical residues that result in enhanced function as measured by binding to C3b, inhibition of the alternative pathway of complement activation, and cofactor activity. In addition, we also demonstrate that despite the enhanced activity of the variola virus protein, its cofactor activity in the factor I-mediated degradation of C3b does not result in the cleavage of the α′ chain of C3b between residues 954–955. Our data have important implications in our understanding of how regulators of complement activation interact with complement, the regulation of the innate immune system, and the rational design of potent complement inhibitors that might be used as therapeutic agents.
PMCID: PMC4138803  PMID: 15699145
4.  Insights into the mechanism of C5aR inhibition by PMX53 via implicit solvent molecular dynamics simulations and docking 
BMC Biophysics  2014;7:5.
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.
doi:10.1186/2046-1682-7-5
PMCID: PMC4141665  PMID: 25170421
Class A GPCR; C5aR; C5a; Complement system; Molecular dynamics; Docking; Implicit solvent; Membrane protein
5.  Molecular Dynamics in Drug Design: New Generations of Compstatin Analogs 
Chemical biology & drug design  2012;79(5):703-718.
We report the computational and rational design of new generations of several tryptophan-rich peptides from the compstatin family. The binding efficacy of the peptides has been tested using extensive molecular dynamics-based structural and physicochemical analysis, using 32 atomic-detail trajectories in explicit water for 22 peptides bound to human, rat, or mouse target protein C3, to a total of 257 nanoseconds. The criteria for the new designs are: (i) optimization for high binding affinity and for the balance between hydrophobicity and polarity to improve solubility compared to known compstatin analogs; and (ii) development of dual specificity anti–human-rat/mouse C3 analogs, which is important for use in animal models for disease, given the species specificity of known compstatin analogs. Three of the new analogs have been analyzed in more detail as they possess strong and novel binding characteristics and are promising candidates for further optimization. This work paves the way for the development of an improved therapeutic for age-related macular degeneration, and other complement system-mediated diseases, compared to known compstatin variants.
doi:10.1111/j.1747-0285.2012.01324.x
PMCID: PMC3319835  PMID: 22233517
6.  The Two Sides of Complement C3d: Evolution of Electrostatics in a Link between Innate and Adaptive Immunity 
PLoS Computational Biology  2012;8(12):e1002840.
The interaction between complement fragment C3d and complement receptor 2 (CR2) is a key aspect of complement immune system activation, and is a component in a link between innate and adaptive immunities. The complement immune system is an ancient mechanism for defense, and can be found in species that have been on Earth for the last 600 million years. However, the link between the complement system and adaptive immunity, which is formed through the association of the B-cell co-receptor complex, including the C3d-CR2 interaction, is a much more recent adaptation. Human C3d and CR2 have net charges of −1 and +7 respectively, and are believed to have evolved favoring the role of electrostatics in their functions. To investigate the role of electrostatics in the function and evolution of human C3d and CR2, we have applied electrostatic similarity methods to identify regions of evolutionarily conserved electrostatic potential based on 24 homologues of complement C3d and 4 homologues of CR2. We also examine the effects of structural perturbation, as introduced through molecular dynamics and mutations, on spatial distributions of electrostatic potential to identify perturbation resistant regions, generated by so-called electrostatic “hot-spots”. Distributions of electrostatic similarity based on families of perturbed structures illustrate the presence of electrostatic “hot-spots” at the two functional sites of C3d, while the surface of CR2 lacks electrostatic “hot-spots” despite its excessively positive nature. We propose that the electrostatic “hot-spots” of C3d have evolved to optimize its dual-functionality (covalently attaching to pathogen surfaces and interaction with CR2), which are both necessary for the formation B-cell co-receptor complexes. Comparison of the perturbation resistance of the electrostatic character of the homologues of C3d suggests that there was an emergence of a new role of electrostatics, and a transition in the function of C3d, after the divergence of jawless fish.
Author Summary
Complement fragment C3d is a thioester-containing protein that is a key component/domain in the complement system, an ancient line of defense, due to its ability to covalently attach to pathogen cell surfaces, such as bacteria. As the immune system evolved in complexity, from acellular defense mechanisms to multicellular systems with memory, so has the function of C3d. In humans, but not lower species such as invertebrates, C3d attached to pathogen surfaces binds B-cell co-receptor CR2, in conjunction with an antibody/antigen complex, forming a link between the innate and adaptive immune systems. The C3d-CR2 interaction ultimately increases B-cell sensitivity to the C3d tagged pathogen by 1,000–10,000 fold, and is known to be driven by electrostatic forces. Since electrostatics are crucial to the C3d-CR2 interaction, it is likely that probing the evolution of the electrostatics of C3d and CR2 will provide insight into this gained function. To this end, we employ a novel computational approach for identifying the electrostatic “hot-spots” of C3d and CR2, which are produced by clusters of like-charged residues found on the surface of the protein. Electrostatic “hot-spots” are often evolutionarily favored and in this study provide new insight into the evolution of C3d in its role in a link between innate and adaptive immunity.
doi:10.1371/journal.pcbi.1002840
PMCID: PMC3531323  PMID: 23300422
7.  Insights into the Structure, Correlated Motions, and Electrostatic Properties of Two HIV-1 gp120 V3 Loops 
PLoS ONE  2012;7(11):e49925.
The V3 loop of the glycoprotein 120 (gp120) is a contact point for cell entry of HIV-1 leading to infection. Despite sequence variability and lack of specific structure, the highly flexible V3 loop possesses a well-defined role in recognizing and selecting cell-bound coreceptors CCR5 and CXCR4 through a mechanism of charge complementarity. We have performed two independent molecular dynamics (MD) simulations to gain insights into the dynamic character of two V3 loops with slightly different sequences, but significantly different starting crystallographic structures. We have identified highly populated trajectory-specific salt bridges between oppositely charged stem residues Arg9 and Glu25 or Asp29. The two trajectories share nearly identical correlated motions within the simulations, despite their different overall structures. High occupancy salt bridges play a key role in the major cross-correlated motions in both trajectories, and may be responsible for transient structural stability in preparation for coreceptor binding. In addition, the two V3 loops visit conformations with similarities in spatial distributions of electrostatic potentials, despite their inherent flexibility, which may play a role in coreceptor recognition. It is plausible that cooperativity between overall electrostatic potential, charged residue interactions, and correlated motions could be associated with a coreceptor selection and binding.
doi:10.1371/journal.pone.0049925
PMCID: PMC3501474  PMID: 23185486
8.  Design of a Modified Mouse Protein with Ligand Binding Properties of its Human Analog by Molecular Dynamics Simulations: The Case of C3 Inhibition by Compstatin 
Proteins  2011;79(11):3166-3179.
The peptide compstatin and its derivatives inhibit the complement-component protein C3 in primate mammals and are potential therapeutic agents against unregulated activation of complement in humans, but are inactive against C3 from lower mammals. Recent molecular dynamics (MD) simulations showed that the most potent compstatin analog comprised entirely of natural amino acids (W4A9) had a smaller affinity for rat C3, due to reproducible changes in the rat protein structure with respect to the human protein, which eliminated or weakened specific protein-ligand interactions seen in the human C3:W4A9 complex. Here, we study by MD simulations three W4A9 complexes with the mouse C3 protein, and two “transgenic” mouse derivatives, containing a small number (6-9) of human C3 substitutions. The mouse complex experiences the conformational changes and affinity reduction of the rat complex. In the “transgenic” complexes the conformation remains closer to that of the human complex, the protein-ligand interactions are improved, and the affinity for compstatin becomes “human-like”. The present work creates new avenues for a compstatin-sensitive animal model. A similar strategy, involving the comparison of a series of complexes by MD simulations, could be used to design “transgenic” sequences in other systems.
doi:10.1002/prot.23149
PMCID: PMC3193182  PMID: 21989937
Innate immune response; Complement system; C3 inhibitors; Compstatin analogs; Molecular Dynamics
9.  A New Generation of Potent Complement Inhibitors of the Compstatin Family 
Chemical biology & drug design  2011;77(6):431-440.
Compstatin family peptides are potent inhibitors of the complement system and promising drug candidates against diseases involving under-regulated complement activation. Compstatin is a 13-residue cyclized peptide that inhibits cleavage of complement protein C3, preventing downstream complement activation. We present three new compstatin variants, characterized by tryptophan replacement at positions 1 and/or 13. Peptide design was based on physicochemical reasoning and was inspired by earlier work which identified tryptophan substitutions at positions 1 and 13 in peptides with predicted C3c binding abilities (Bellows, M. L.; Fung, H. K.; Taylor, M. S.; Floudas, C. A.; López de Victoria, A.; Morikis, D. (2010) Biophys J 98: 2337–2346). The new variants preserve distinct polar and nonpolar surfaces of compstatin, but have altered local interaction capabilities with C3. All three peptides exhibited potent C3 binding by surface plasmon resonance (SPR) and potent complement inhibition by ELISAs. We also present ELISA data and detailed SPR kinetic data of three peptides from previous computational design.
doi:10.1111/j.1747-0285.2011.01111.x
PMCID: PMC3095715  PMID: 21352502
Structure-Based Drug Design; Peptide Design; Compstatin Family; Complement System; Complement System Inhibitors
10.  Clustering of HIV-1 Subtypes Based on gp120 V3 Loop electrostatic properties 
BMC Biophysics  2012;5:3.
Background
The V3 loop of the glycoprotein gp120 of HIV-1 plays an important role in viral entry into cells by utilizing as coreceptor CCR5 or CXCR4, and is implicated in the phenotypic tropisms of HIV viruses. It has been hypothesized that the interaction between the V3 loop and CCR5 or CXCR4 is mediated by electrostatics. We have performed hierarchical clustering analysis of the spatial distributions of electrostatic potentials and charges of V3 loop structures containing consensus sequences of HIV-1 subtypes.
Results
Although the majority of consensus sequences have a net charge of +3, the spatial distribution of their electrostatic potentials and charges may be a discriminating factor for binding and infectivity. This is demonstrated by the formation of several small subclusters, within major clusters, which indicates common origin but distinct spatial details of electrostatic properties. Some of this information may be present, in a coarse manner, in clustering of sequences, but the spatial details are largely lost. We show the effect of ionic strength on clustering of electrostatic potentials, information that is not present in clustering of charges or sequences. We also make correlations between clustering of electrostatic potentials and net charge, coreceptor selectivity, global prevalence, and geographic distribution. Finally, we interpret coreceptor selectivity based on the N6X7T8|S8X9 sequence glycosylation motif, the specific positive charge location according to the 11/24/25 rule, and the overall charge and electrostatic potential distribution.
Conclusions
We propose that in addition to the sequence and the net charge of the V3 loop of each subtype, the spatial distributions of electrostatic potentials and charges may also be important factors for receptor recognition and binding and subsequent viral entry into cells. This implies that the overall electrostatic potential is responsible for long-range recognition of the V3 loop with coreceptors CCR5/CXCR4, whereas the charge distribution contributes to the specific short-range interactions responsible for the formation of the bound complex. We also propose a scheme for coreceptor selectivity based on the sequence glycosylation motif, the 11/24/25 rule, and net charge.
doi:10.1186/2046-1682-5-3
PMCID: PMC3295656  PMID: 22313935
HIV-1; protein-receptor interactions; Poisson-Boltzmann electrostatics; electrostatic similarity distance; electrostatic clustering
11.  Species specificity of the complement inhibitor compstatin investigated by all-atom molecular dynamics simulations 
Proteins  2010;78(12):2655-2667.
The development of compounds to regulate the activation of the complement system in non-primate species is of profound interest because it can provide models for human diseases. The peptide compstatin inhibits protein C3 in primate mammals and is a potential therapeutic agent against unregulated activation of complement in humans but is inactive against nonprimate species. Here, we elucidate this species specificity of compstatin by molecular dynamics simulations of complexes between the most potent natural compstatin analog and human or rat C3. The results are compared against an experimental conformation of the human complex, determined recently by X-ray diffraction at 2.4-Å resolution. The human complex simulations provide information on the relative contributions to stability of specific C3 and compstatin residues. In the rat simulations, the protein undergoes reproducible conformational changes, which eliminate or weaken specific interactions and reduce the complex stability. The simulation insights can be used to design improved compstatin-based inhibitors for human C3 and active inhibitors against lower mammals.
doi:10.1002/prot.22780
PMCID: PMC3138065  PMID: 20589629
molecular dynamics simulations; compstatin analogs; C3 inhibitors; complement system; innate immune response
12.  Contribution of Specific Amino Acid Changes in Penicillin Binding Protein 1 to Amoxicillin Resistance in Clinical Helicobacter pylori isolates ▿  
Amoxicillin is commonly used to treat Helicobacter pylori, a major cause of peptic ulcers, stomach cancer, and B-cell mucosa-associated lymphoid tissue lymphoma. Amoxicillin resistance in H. pylori is increasing steadily, especially in developing countries, leading to treatment failures. In this study, we characterize the mechanism of amoxicillin resistance in the U.S. clinical isolate B258. Transformation of amoxicillin-susceptible strain 26695 with the penicillin binding protein 1 gene (pbp1) from B258 increased the amoxicillin resistance of 26695 to equal that of B258, while studies using biotinylated amoxicillin showed a decrease in the binding of amoxicillin to the PBP1 of B258. Transformation with 4 pbp1 fragments, each encompassing several amino acid substitutions, combined with site-directed mutagenesis studies, identified 3 amino acid substitutions in PBP1 of B258 which affected amoxicillin susceptibility (Val 469 Met, Phe 473 Leu, and Ser 543 Arg). Homology modeling showed the spatial orientation of these specific amino acid changes in PBP1 from 26695 and B258. The results of these studies demonstrate that amoxicillin resistance in the clinical U.S. isolate B258 is due solely to an altered PBP1 protein with a lower binding affinity for amoxicillin. Homology modeling analyses using previously identified amino acid substitutions of amoxicillin-resistant PBP1s demonstrate the importance of specific amino acid substitutions in PBP1 that affect the binding of amoxicillin in the putative binding cleft, defining those substitutions deemed most important in amoxicillin resistance.
doi:10.1128/AAC.00545-10
PMCID: PMC3019683  PMID: 20956585
13.  Electrostatic Clustering and Free Energy Calculations Provide a Foundation for Protein Design and Optimization 
Annals of Biomedical Engineering  2010;39(4):1252-1263.
Electrostatic interactions are ubiquitous in proteins and dictate stability and function. In this review, we discuss several methods for the analysis of electrostatics in protein–protein interactions. We discuss alanine-scanning mutagenesis, Poisson–Boltzmann electrostatics, free energy calculations, electrostatic similarity distances, and hierarchical clustering of electrostatic potentials. Our recently developed computational framework, known as Analysis of Electrostatic Similarities Of Proteins (AESOP), incorporates these tools to efficiently elucidate the role of electrostatic potentials in protein interactions. We present the application of AESOP to several proteins and protein complexes, for which charge is purported to facilitate protein association. Specifically, we illustrate how recent work has shaped the formulation of electrostatic calculations, the correlation of electrostatic free energies and electrostatic potential clustering results with experimental binding and activity data, the pH dependence of protein stability and association, the design of mutant proteins with enhanced immunological activity, and how AESOP can expose deficiencies in structural models and experimental data. This integrative approach can be utilized to develop mechanistic models and to guide experimental studies by predicting mutations with desired physicochemical properties and function. Alteration of the electrostatic properties of proteins offers a basis for the design of proteins with optimized binding and activity.
doi:10.1007/s10439-010-0226-9
PMCID: PMC3069318  PMID: 21140293
Alanine scan; Poisson–Boltzmann equation; Continuum electrostatics; Solvation; Electrostatic potential; Hierarchical clustering
14.  A multifaceted study of stigma/style cysteine-rich adhesin (SCA)-like Arabidopsis lipid transfer proteins (LTPs) suggests diversified roles for these LTPs in plant growth and reproduction 
Journal of Experimental Botany  2010;61(15):4277-4290.
Lily stigma/style cysteine-rich adhesin (SCA), a plant lipid transfer protein (LTP) which is secreted into the extracellular matrix, functions in pollen tube guidance in fertilization. A gain-of-function mutant (ltp5-1) for Arabidopsis LTP5, an SCA-like molecule, was recently shown to display defects in sexual reproduction. In the current study, it is reported that ltp5-1 plants have dwarfed primary shoots, delayed hypocotyl elongation, various abnormal tissue fusions, and display multibranching. These mutant phenotypes in vegetative growth are recessive. No abnormality was found in ltp5-1/+ plants. In a phylogenetic analysis of plant LTPs, SCA-like Arabidopsis LTPs were classified with conventional plant LTPs. Homology modelling-based electrostatic similarity index (ESI) clustering was used to show diversity in spatial distributions of electrostatic potentials of SCA-like LTPs, suggestive of their various roles in interaction in the extracellular matrix space. β-Glucuronidase (GUS) analysis showed that SCA-like Arabidopsis LTP genes are diversely present in various tissues. LTP4 was found specifically in the guard cells and LTP6 in trichomes as well as in other tissues. LTP1 levels were specifically abundant in the stigma, and both LTP3 and LTP6 in the ovules. LTP2 and LTP4 gene levels were up-regulated in whole seedlings with 20% polyethylene glycol (PEG) and 300 mM NaCl treatments, respectively. LTP5 was up-regulated in the hypocotyl with 3 d dark growth conditions. LTP6 was specifically expressed in the tip of the cotyledon under drought stress conditions. The results suggest that SCA-like Arabidopsis LTPs are multifunctional, with diversified roles in plant growth and reproduction.
doi:10.1093/jxb/erq228
PMCID: PMC2955742  PMID: 20667964
Arabidopsis thaliana; electrostatics; extracellular matrix (ECM); lipid transfer protein (LTP); small secreted peptide; stigma/style cysteine-rich adhesin (SCA)

Results 1-14 (14)