Assessing Protein-Protein Docking Protocols: Case Studies of G-Protein-Coupled Receptor Interactions.

The interactions of G-protein-coupled receptors (GPCRs) with other proteins are critical in several cellular processes but resolving their structural dynamics remains challenging. An increasing number of GPCR complexes have been experimentally resolved but others including receptor variants are yet to be characterized, necessitating computational predictions of their interactions. Although integrative approaches with multi-scale simulations would provide rigorous estimates of their conformational dynamics, protein-protein docking remains a first tool of choice of many researchers due to the availability of open-source programs and easy to use web servers with reasonable predictive power. Protein-protein docking algorithms have limited ability to consider protein flexibility, environment effects, and entropy contributions and are usually a first step towards more integrative approaches. The two critical steps of docking: the sampling and scoring algorithms have improved considerably and their performance has been validated against experimental data. In this chapter, we provide an overview and generalized protocol of a few docking protocols using GPCRs as test cases. In particular, we demonstrate the interactions of GPCRs with extracellular protein ligands and an intracellular protein effectors (G-protein) predicted from docking approaches and test their limitations. The current chapter will help researchers critically assess docking protocols and predict experimentally testable structures of GPCR complexes.

Insights into the dynamic interactions at chemokine-receptor interfaces and mechanistic models of chemokine binding.

Chemokine receptors are the central signaling hubs of several processes such as cell migration, chemotaxis and cell positioning. In this graphical review, we provide an overview of the structural and mechanistic principles governing chemokine recognition that are currently emerging. Structural models of chemokine-receptor co-complexes with endogenous chemokines, viral chemokines and therapeutics have been resolved that highlight multiple interaction sites, termed as CRS1, CRS1.5 etc. The first site of interaction has been shown to be the N-terminal domain of the receptors (CRS1 site). A large structural flexibility of the N-terminal domain has been reported that was supported by both experimental and simulation studies. Upon chemokine binding, the N-terminal domain appears to show constricted dynamics and opens up to interact with the chemokine via a large interface. The subsequent sites such as CRS1.5 and CRS2 sites have been structurally well resolved although differences arise such as the localization of the N-terminus of the ligand to a major or minor pocket of the orthosteric binding site. Several computational studies have highlighted the dynamic protein-protein interface at the CRS1 site that seemingly appears to resolve the differences in NMR and mutagenesis studies. Interestingly, the differential dynamics at the CRS1 site suggests a mixed model of binding with complex signatures of both conformational selection and induced fit models. Integrative experimental and computational approaches could help unravel the structural basis of promiscuity and specificity in chemokine-receptor binding and open up new avenues of therapeutic design.

Allosteric modulation of the chemokine receptor-chemokine CXCR4-CXCL12 complex by tyrosine sulfation.

The chemokine receptor CXCR4 and its cognate ligand CXCL12 mediate pathways that lead to cell migration and chemotaxis. Although the structural details of related receptor-ligand complexes have been resolved, the roles of the N-terminal domain of the receptor and post-translational sulfation that are determinants of ligand selectivity and affinity remain unclear. Here, we analyze the structural dynamics induced by receptor sulfation by combining molecular dynamics, docking and network analysis. The sulfotyrosine residues, 7YsN-term, 12YsN-term and 21YsN-term allow the N-terminal domain of the apo-sulfated receptor to adopt an "open" conformation that appears to facilitate ligand binding. The overall topology of the CXCR4-CXCL12 complex is independent of the sulfation state, but an extensive network of protein-protein interactions characterizes the sulfated receptor, in line with its increased ligand affinity. The altered interactions of sulfotyrosine residues, such as 21YsN-term-47RCXCL12 replacing the 21YN-term-13FCXCL12 interaction, propagate via allosteric pathways towards the receptor lumen. In particular, our results suggest that the experimentally-reported receptor-ligand interactions 262D6.58-8RCXCL12 and 277E7.28-12RCXCL12 could be dependent on the sulfation state of the receptor and need to be carefully analyzed. Our work is an important step in understanding chemokine-receptor interactions and how post-translational modifications could modulate receptor-ligand complexes.

Effect of diversity in gp41 membrane proximal external region of primary HIV-1 Indian subtype C sequences on interaction with broadly neutralizing antibodies 4E10 and 10E8.

Human Immunodeficiency Virus-1 Clade C (HIV-1C) dominates the AIDS epidemic in India, afflicting 2.1 million individuals within the country and more than 15 million people worldwide. Membrane proximal external region (MPER) is an attractive target for broadly neutralizing antibody (bNAb) based therapies. However, information on MPER sequence diversity from India is meagre due to limited sampling of primary viral sequences. In the present study, we examined the variation in MPER of HIV-1C from 24 individuals in Mumbai, India by high throughput sequencing of uncultured viral sequences. Deep sequencing of MPER (662-683; HXB2 envelope amino acid numbering) allowed quantification of intra-individual variation up to 65% at positions 662, 665, 668, 674 and 677 within this region. These variable positions included contact sites targeted by bNAbs 2F5, Z13e1, 4E10 as well as 10E8. Both major and minor epitope variants i.e. 'haplotypes' were generated for each sample dataset. A total of 23, 34 and 25 unique epitope haplotypes could be identified for bNAbs 2F5, Z13e1 and 4E10/10E8 respectively. Further analysis of 4E10 and 10E8 epitopes from our dataset and meta-analysis of previously reported HIV-1 sequences from India revealed 26 epitopes (7 India-specific), heretofore untested for neutralization sensitivity. Peptide-Ab docking predicted 13 of these to be non-binding to 10E8. ELISA, Surface Plasmon Resonance and peptide inhibition of HIV-1 neutralization assays were then performed which validated predicted weak/non-binding interactions for peptides corresponding to six of these epitopes. These results highlight the under-representation of 10E8 non-binding HIV-1C MPER sequences from India. Our study thus underscores the need for increased surveillance of primary circulating envelope sequences for development of efficacious bNAb-based interventions in India.

Revealing the molecular interactions of aptamers that specifically bind to the extracellular domain of HER2 cancer biomarker protein: An in silico assessment.

Single-stranded (ss) oligonucleotide aptamers are emerging as the promising substitutes for monoclonal antibodies because of their low production cost and good batch-to-batch consistency. Aptamers vividly bind to a variety of cellular targets and alter their functions with a remarkable degree of specificities. In this study, the positive clones of human epidermal growth factor receptor 2 (HER2) specific binding ssDNA aptamers which were previously identified by in vitro Systematic Evolution of Ligands by EXponential enrichment (SELEX) process, hitherto lacking the putative binding site information and residues crucial for aptamer recognition are studied. Primarily, four putative DNA binding regions present on the HER2 extracellular domain (ECD) were identified using prediction servers and electrostatic potential maps, which were further exploited to delineate the aptamer binding features. Molecular docking and molecular dynamics (MD) simulations revealed stable binding nature of three aptamers (H2>H1>H6), which chose Site 2a as preferred binding site present on the HER2(ECD) protein. Furthermore, amino acid residues viz. Asn37, Gln59, Arg81-Gln84, Asp88, and Lys128 of Site 2a were found to be crucial for high-affinity binding. This knowledge can be utilized as a benchmark for the future studies, in search for better and highly specific anti-HER2 aptamers as cancer therapeutics or as diagnostic agents.

Protein-Protein Interaction between Surfactant Protein D and DC-SIGN via C-Type Lectin Domain Can Suppress HIV-1 Transfer.

Surfactant protein D (SP-D) is a soluble C-type lectin, belonging to the collectin (collagen-containing calcium-dependent lectin) family, which acts as an innate immune pattern recognition molecule in the lungs at other mucosal surfaces. Immune regulation and surfactant homeostasis are salient functions of SP-D. SP-D can bind to a range of viral, bacterial, and fungal pathogens and trigger clearance mechanisms. SP-D binds to gp120, the envelope protein expressed on HIV-1, through its C-type lectin or carbohydrate recognition domain. This is of importance since SP-D is secreted by human mucosal epithelial cells and is present in the female reproductive tract, including vagina. Another C-type lectin, dendritic cell (DC)-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN), present on the surface of the DCs, also binds to HIV-1 gp120 and facilitates viral transfer to the lymphoid tissues. DCs are also present at the site of HIV-1 entry, embedded in vaginal or rectal mucosa. In the present study, we report a direct protein-protein interaction between recombinant forms of SP-D (rfhSP-D) and DC-SIGN via their C-type lectin domains. Both SP-D and DC-SIGN competed for binding to immobilized HIV-1 gp120. Pre-incubation of human embryonic kidney cells expressing surface DC-SIGN with rfhSP-D significantly inhibited the HIV-1 transfer to activated peripheral blood mononuclear cells. In silico analysis revealed that SP-D and gp120 may occupy same sites on DC-SIGN, which may explain the reduced transfer of HIV-1. In summary, we demonstrate, for the first time, that DC-SIGN is a novel binding partner of SP-D, and this interaction can modulate HIV-1 capture and transfer to CD4+ T cells. In addition, the present study also reveals a novel and distinct mechanism of host defense by SP-D against HIV-1.

Evaluation of 4-thiazolidinone derivatives as potential reverse transcriptase inhibitors against HIV-1 drug resistant strains.

Rapid emergence of drug resistance is crucial in management of HIV infection limiting implementation of efficacious drugs in the ART regimen. Designing new molecules against HIV drug resistant strains is utmost essential. Based on the anti-HIV-1 activity, we selected four 4-thiazolidinone derivatives (S009-1908, S009-1909, S009-1911, S009-1912) and studied their interaction with reverse transcriptase (RT) from a panel of 10 clinical isolates (8 nevirapine resistant and two susceptible) using in silico methods, and inhibition pattern using in vitro cell based assays. On the basis of binding affinity observed in in silico analysis, 2-(2-chloro-6-nitrophenyl)-3-(4, 6-dimethylpyridin-2-yl) thiazolidin-4-one (S009-1912) was identified as the lead molecule followed by S009-1908, S009-1909 and S009-1911. The in vitro activity against the same panel was assessed using TZM-bl assay (IC50: 0.4-11.44µg/ml, TI: 4-126) and subsequently in PBMC assay against a nevirapine resistant clinical isolate (IC50: 0.8-6.65µg/ml, TI: 8.31-11.43) and standard strain from NIH ARRRP (IC50: 0.95-3.6µg/ml, TI: 9-26). The study shows analogue with pyrimidin-2-yl amino substitution at N-3 position of thiazolidin-4-one ring (S009-1908, S009-1909, S009-1911) exhibited enhanced activity as compared to pyridin-2-yl substituted derivatives (S009-1912), suggesting the use 4-thiazolidinones for developing potent inhibitors against HIV-1 drug resistant strains.

Cervical cancer in Indian women reveals contrasting association among common sub-family of HLA class I alleles.

We studied the relationship between human leukocyte antigen (HLA) class I alleles and cervical cancer among Indian women. Seventy-five cervical cancer cases were compared with 175 noncancer controls. Cervical biopsy tissue specimen from cancer cases and cervical swab specimen from controls were collected for HPV detection and typing. Blood was taken for HLA typing by PCR-SSOP method. The impact of HLA class I alleles on cervical cancer risk was evaluated using StatCalc program (Epi Info version 6.0.4. CDC Atlanta, GA, USA), and confirmed with Bonferroni correction. Results revealed HLA-B*37, HLA-B*58 were associated significantly with increased risk while HLA-B*40 with decreased risk for cervical cancer. At high-resolution analysis after Bonferroni correction, HLA-B*37:01 allele was associated with increased risk, whereas HLA-B*40:06 was with decreased risk for cervical cancer. HLA-B*37:01 and HLA-B*40:06 belong to the same superfamily of HLA-B44. In silico analysis revealed different binding affinities of HLA-B*37:01 and HLA-B*40:06 for the epitopes predicted for E6 and L1 proteins of HPV16. The higher binding affinity of epitopes to B*40:06, as revealed by docking studies, supports the hypothesis that this allele is able to present the antigenic peptides more efficiently than B*37:01 and thereby can protect the carriers from the risk of cervical cancer. Thus, there is a clear indication that HLA plays an important role in the development of cervical cancer in HPV-infected women. Identification of these factors in high-risk HPV-infected women may help in reducing the cervical cancer burden in India.

Surfactant protein D inhibits HIV-1 infection of target cells via interference with gp120-CD4 interaction and modulates pro-inflammatory cytokine production.

Surfactant Protein SP-D, a member of the collectin family, is a pattern recognition protein, secreted by mucosal epithelial cells and has an important role in innate immunity against various pathogens. In this study, we confirm that native human SP-D and a recombinant fragment of human SP-D (rhSP-D) bind to gp120 of HIV-1 and significantly inhibit viral replication in vitro in a calcium and dose-dependent manner. We show, for the first time, that SP-D and rhSP-D act as potent inhibitors of HIV-1 entry in to target cells and block the interaction between CD4 and gp120 in a dose-dependent manner. The rhSP-D-mediated inhibition of viral replication was examined using three clinical isolates of HIV-1 and three target cells: Jurkat T cells, U937 monocytic cells and PBMCs. HIV-1 induced cytokine storm in the three target cells was significantly suppressed by rhSP-D. Phosphorylation of key kinases p38, Erk1/2 and AKT, which contribute to HIV-1 induced immune activation, was significantly reduced in vitro in the presence of rhSP-D. Notably, anti-HIV-1 activity of rhSP-D was retained in the presence of biological fluids such as cervico-vaginal lavage and seminal plasma. Our study illustrates the multi-faceted role of human SP-D against HIV-1 and potential of rhSP-D for immunotherapy to inhibit viral entry and immune activation in acute HIV infection.

HPV16 E6 variants: frequency, association with HPV types and in silico analysis of the identified novel variants.

High-risk human papillomavirus (HPV) types, specifically HPV 16 E6 variants are involved in viral persistence and the development of cervical lesions. India contributes to 1/3rd of the global cervical cancer deaths; however, information on E6 variants in the Indian population is limited. Information on these variants is essential for successful implementation of cervical cancer immunization programs. The E6 variants and their possible biological implications to the outcome of infection were studied in women attending the Tata Memorial Hospital, Mumbai, India. Cervical cancer patients with HPV 16 as a single infection (n = 33), co-infection with another HPV type (n = 20) or with multiple types (n = 10) were examined for HPV16 E6 variants using PCR and sequence analysis. The variants were identified using the prototype sequence (HPV 16R) belonging to the European lineage. The results revealed that the European T350G was the most common variant (50%) followed by the European prototype (40.3%) and the North-American (N = 3; 4.8%). The European prototype was significantly more frequent in patients infected with HPV16 alone (P < 0.05, C.I. 1.2-13.6), while the European T350G variants were seen in women with co-infections. The North-American lineage was found in women infected with HPV16 and 33. Three novel variants were identified of which two were non-synonymous. Phylogenetic analysis revealed that the variant F69L + L83V is not related to any of these lineages, while the variant M137L + L83V is closely related to the North American variant. This study found a difference in the prevalence of E6 variants compared to earlier Indian studies and their association with type of infection.

In silico study on binding specificity of gonadotropins and their receptors: design of a novel and selective peptidomimetic for human follicle stimulating hormone receptor.

Gonadotropins bind to specific receptors in spite of sharing a high level of sequence and structural similarity. This specific binding is crucial for maintaining the reproductive health of an organism. In this study, residues that dictate the receptor binding specificity of the gonadotropins (FSH and LH) have been identified using combination of in silico methods. Docking studies (ZDOCK), based on the systematic replacement of these residues, confirmed its importance in receptor binding. An interesting observation is that the relative positioning of the residues conferring binding specificity varied for the gonadotropin-receptor complexes. This spatial difference of the key residues could be exploited for design of specific modulators. Based on the identified residues, we have rationally designed a peptidomimetic (FSHP) that displays good binding affinity and specificity for hFSHR. FSHP was developed by screening 3.9 million compounds using pharmacophore-shape similarity followed by fragment-based approach. It was observed that FSHP and hFSHâ can share the same receptor binding site thereby mimicking the native hFSHR-FSH interactions. FSHP also displayed higher binding affinity to hFSHR as compared to two reported hFSHR antagonists. MD simulation studies on hFSHR-FSHP complex revealed that FSHP is conformationally rigid and the intermolecular interactions are maintained during the course of simulation.

Correction: In Silico Study on Binding Specificity of Gonadotropins and Their Receptors: Design of a Novel and Selective Peptidomimetic for Human Follicle Stimulating Hormone Receptor.

[This corrects the article DOI: 10.1371/journal.pone.0064475.].