Anthony E. Getschman

Sulfopeptide probes of the CXCR4/CXCL12 interface reveal oligomer-specific contacts and chemokine allostery.

Tyrosine sulfation is a post-translational modification that enhances protein-protein interactions and may identify druggable sites in the extracellular space. The G protein-coupled receptor CXCR4 is a prototypical example with three potential sulfation sites at positions 7, 12, and 21. Each receptor sulfotyrosine participates in specific contacts with its chemokine ligand in the structure of a soluble, dimeric CXCL12:CXCR4(1-38) complex, but their relative importance for CXCR4 binding and activation by the monomeric chemokine remains undefined. NMR titrations with short sulfopeptides showed that the tyrosine motifs of CXCR4 varied widely in their contributions to CXCL12 binding affinity and site specificity. Whereas the Tyr21 sulfopeptide bound the same site as in previously solved structures, the Tyr7 and Tyr12 sulfopeptides interacted nonspecifically. Surprisingly, the unsulfated Tyr7 peptide occupied a hydrophobic site on the CXCL12 monomer that is inaccessible in the CXCL12 dimer. Functional analysis of CXCR4 mutants validated the relative importance of individual CXCR4 sulfotyrosine modifications (Tyr21 > Tyr12 > Tyr7) for CXCL12 binding and receptor activation. Biophysical measurements also revealed a cooperative relationship between sulfopeptide binding at the Tyr21 site and CXCL12 dimerization, the first example of allosteric behavior in a chemokine. Future ligands that occupy the sTyr21 recognition site may act as both competitive inhibitors of receptor binding and allosteric modulators of chemokine function. Together, our data suggests that sulfation does not ubiquitously enhance complex affinity and that distinct patterns of tyrosine sulfation could encode oligomer selectivity, implying another layer of regulation for chemokine signaling.

New paradigms in chemokine receptor signal transduction: Moving beyond the two-site model

Chemokine receptor (CKR) signaling forms the basis of essential immune cellular functions, and dysregulated CKR signaling underpins numerous disease processes of the immune system and beyond. CKRs, which belong to the seven transmembrane domain receptor (7TMR) superfamily, initiate signaling upon binding of endogenous, secreted chemokine ligands. Chemokine-CKR interactions are traditionally described by a two-step/two-site mechanism, in which the CKR N-terminus recognizes the chemokine globular core (i.e. site 1 interaction), followed by activation when the unstructured chemokine N-terminus is inserted into the receptor TM bundle (i.e. site 2 interaction). Several recent studies challenge the structural independence of sites 1 and 2 by demonstrating physical and allosteric links between these supposedly separate sites. Others contest the functional independence of these sites, identifying nuanced roles for site 1 and other interactions in CKR activation. These developments emerge within a rapidly changing landscape in which CKR signaling is influenced by receptor PTMs, chemokine and CKR dimerization, and endogenous non-chemokine ligands. Simultaneous advances in the structural and functional characterization of 7TMR biased signaling have altered how we understand promiscuous chemokine-CKR interactions. In this review, we explore new paradigms in CKR signal transduction by considering studies that depict a more intricate architecture governing the consequences of chemokine-CKR interactions.

Structure-Based Identification of Novel Ligands Targeting Multiple Sites within a Chemokine–G-Protein-Coupled-Receptor Interface

CXCL12 is a human chemokine that recognizes the CXCR4 receptor and is involved in immune responses and metastatic cancer. Interactions between CXCL12 and CXCR4 are an important drug target but, like other elongated protein-protein interfaces, present challenges for small molecule ligand discovery due to the relatively shallow and featureless binding surfaces. Calculations using an NMR complex structure revealed a binding hot spot on CXCL12 that normally interacts with the I4/I6 residues from CXCR4. Virtual screening was performed against the NMR model, and subsequent testing has verified the specific binding of multiple docking hits to this site. Together with our previous results targeting two other binding pockets that recognize sulfotyrosine residues (sY12 and sY21) of CXCR4, including a new analog against the sY12 binding site reported herein, we demonstrate that protein-protein interfaces can often possess multiple sites for engineering specific small molecule ligands that provide lead compounds for subsequent optimization by fragment based approaches.

Pancreatic Cancer Cell Migration and Metastasis Is Regulated by Chemokine-Biased Agonism and Bioenergetic Signaling

Patients with pancreatic ductal adenocarcinoma (PDAC) invariably succumb to metastatic disease, but the underlying mechanisms that regulate PDAC cell movement and metastasis remain little understood. In this study, we investigated the effects of the chemokine gene CXCL12, which is silenced in PDAC tumors, yet is sufficient to suppress growth and metastasis when re-expressed. Chemokines like CXCL12 regulate cell movement in a biphasic pattern, with peak migration typically in the low nanomolar concentration range. Herein, we tested the hypothesis that the biphasic cell migration pattern induced by CXCL12 reflected a biased agonist bioenergetic signaling that might be exploited to interfere with PDAC metastasis. In human and murine PDAC cell models, we observed that nonmigratory doses of CXCL12 were sufficient to decrease oxidative phosphorylation and glycolytic capacity and to increase levels of phosphorylated forms of the master metabolic kinase AMPK. Those same doses of CXCL12 locked myosin light chain into a phosphorylated state, thereby decreasing F-actin polymerization and preventing cell migration in a manner dependent upon AMPK and the calcium-dependent kinase CAMKII. Notably, at elevated concentrations of CXCL12 that were insufficient to trigger chemotaxis of PDAC cells, AMPK blockade resulted in increased cell movement. In two preclinical mouse models of PDAC, administration of CXCL12 decreased tumor dissemination, supporting our hypothesis that chemokine-biased agonist signaling may offer a useful therapeutic strategy. Our results offer a mechanistic rationale for further investigation of CXCL12 as a potential therapy to prevent or treat PDAC metastasis.

Structural Analysis of a Novel Small Molecule Ligand Bound to the CXCL12 Chemokine.

CXCL12 binds to CXCR4, promoting both chemotaxis of lymphocytes and metastasis of cancer cells. We previously identified small molecule ligands that bind CXCL12 and block CXCR4-mediated chemotaxis. We now report a 1.9 Å resolution X-ray structure of CXCL12 bound by such a molecule at a site normally bound by sY21 of CXCR4. The complex structure reveals binding hot spots for future inhibitor design and suggests a new approach to targeting CXCL12–CXCR4 signaling in drug discovery.

Protein engineering of the chemokine CCL20 prevents psoriasiform dermatitis in an IL-23–dependent murine model

Significance Psoriasis is a chronic skin disease characterized by the infiltration of inflammatory T cells to the skin in response to injury. When inflammatory T cells and dendritic cells are recruited to the skin by CCL20 and other chemokines, they release cytokines that contribute to psoriatic inflammation. We engineered a molecule derived from the natural CCL20 protein that adopts a unique dimeric structure, partially activates its G-protein receptor, blocks T cell homing, and prevents the signs of psoriasis in a mouse model of this common human skin disease. Our remarkable findings reveal the potential of engineered-CCL20 molecules as therapeutic agents for psoriasis and the general utility of chemokine engineering for treating inflammatory diseases. Psoriasis is a chronic inflammatory skin disease characterized by the infiltration of T cell and other immune cells to the skin in response to injury or autoantigens. Conventional, as well as unconventional, γδ T cells are recruited to the dermis and epidermis by CCL20 and other chemokines. Together with its receptor CCR6, CCL20 plays a critical role in the development of psoriasiform dermatitis in mouse models. We screened a panel of CCL20 variants designed to form dimers stabilized by intermolecular disulfide bonds. A single-atom substitution yielded a CCL20 variant (CCL20 S64C) that acted as a partial agonist for the chemokine receptor CCR6. CCL20 S64C bound CCR6 and induced intracellular calcium release, consistent with G-protein activation, but exhibited minimal chemotactic activity. Instead, CCL20 S64C inhibited CCR6-mediated T cell migration with nominal impact on other chemokine receptor signaling. When given in an IL-23–dependent mouse model for psoriasis, CCL20 S64C prevented psoriatic inflammation and the up-regulation of IL-17A and IL-22. Our results validate CCR6 as a tractable therapeutic target for psoriasis and demonstrate the value of CCL20 S64C as a lead compound.

Exploiting agonist biased signaling of chemokines to target cancer

As knowledge of growth‐independent functions of cancer cells is expanding, exploration into the role of chemokines in modulating cancer pathogenesis, particularly metastasis, continues to develop. However, more study into the mechanisms whereby chemokines direct the migration of cancer cells is needed before specific therapies can be generated to target metastasis. Herein, we draw attention to the longstanding conundrum in the field of chemokine biology that chemokines stimulate migration in a biphasic manner; and explore this phenomenons impact on chemokine function in the context of cancer. Typically, low concentrations of chemokines lead to chemotactic migration and higher concentrations halt migration. The signaling mechanisms that govern this phenomenon remain unclear. Over the last decade, we have defined a novel signaling mechanism for regulation of chemokine migration through ligand oligomerization and biased agonist signaling. We provide insight into this new paradigm for chemokine signaling and discuss how it will impact future exploration into chemokine function and biology. In the pursuit of producing more novel cancer therapies, we suggest a framework for pharmaceutical application of the principles of chemokine oligomerization and biased agonist signaling in cancer.

CCR7 Sulfotyrosine Enhances CCL21 Binding

Chemokines are secreted proteins that direct the migration of immune cells and are involved in numerous disease states. For example, CCL21 (CC chemokine ligand 21) and CCL19 (CC chemokine ligand 19) recruit antigen-presenting dendritic cells and naïve T-cells to the lymph nodes and are thought to play a role in lymph node metastasis of CCR7 (CC chemokine receptor 7)-expressing cancer cells. For many chemokine receptors, N-terminal posttranslational modifications, particularly the sulfation of tyrosine residues, increases the affinity for chemokine ligands and may contribute to receptor ligand bias. Chemokine sulfotyrosine (sY) binding sites are also potential targets for drug development. In light of the structural similarity between sulfotyrosine and phosphotyrosine (pY), the interactions of CCL21 with peptide fragments of CCR7 containing tyrosine, pY, or sY were compared using protein NMR (nuclear magnetic resonance) spectroscopy in this study. Various N-terminal CCR7 peptides maintain binding site specificity with Y8-, pY8-, or sY8-containing peptides binding near the α-helix, while Y17-, pY17-, and sY17-containing peptides bind near the N-loop and β3-stand of CCL21. All modified CCR7 peptides showed enhanced binding affinity to CCL21, with sY having the largest effect.

Differences in Sulfotyrosine Binding amongst CXCR1 and CXCR2 Chemokine Ligands

Tyrosine sulfation, a post-translational modification found on many chemokine receptors, typically increases receptor affinity for the chemokine ligand. A previous bioinformatics analysis suggested that a sulfotyrosine (sY)-binding site on the surface of the chemokine CXCL12 may be conserved throughout the chemokine family. However, the extent to which receptor tyrosine sulfation contributes to chemokine binding has been examined in only a few instances. Computational solvent mapping correctly identified the conserved sulfotyrosine-binding sites on CXCL12 and CCL21 detected by nuclear magnetic resonance (NMR) spectroscopy, demonstrating its utility for hot spot analysis in the chemokine family. In this study, we analyzed five chemokines that bind to CXCR2, a subset of which also bind to CXCR1, to identify hot spots that could participate in receptor binding. A cleft containing the predicted sulfotyrosine-binding pocket was identified as a principal hot spot for ligand binding on the structures of CXCL1, CXCL2, CXCL7, and CXCL8, but not CXCL5. Sulfotyrosine titrations monitored via NMR spectroscopy showed specific binding to CXCL8, but not to CXCL5, which is consistent with the predictions from the computational solvent mapping. The lack of CXCL5–sulfotyrosine interaction and the presence of CXCL8–sulfotyrosine binding suggests a role for receptor post-translational modifications regulating ligand selectivity.

Mutational analysis of CCL20 reveals flexibility of N‐terminal amino acid composition and length

Chemokine–chemokine receptor (CKR) interactions are traditionally described by a two‐step/two‐site mechanism that details the major contact points between chemokine ligands and CKRs leading to ligand recognition and receptor activation. Chemokine recognition site 1 (CRS1) encompasses interactions between the CKR N‐terminus and the globular chemokine core. Chemokine recognition site 2 (CRS2) includes interactions between the unstructured chemokine N‐terminus and the binding pocket of the receptor. The two‐step/two‐site paradigm has been an adequate framework to study the intricacies of chemokine:CKR interactions, but emerging studies highlight the limitations of this model. Here, we present studies of CRS2 interactions between the chemokine CCL20 and its cognate receptor CCR6 driven by the hypothesis that CCL20 interacts with CCR6 as described by the two‐step/two‐site model. CCL20 is a chemokine with an unusually short N‐terminus of 5 residues (NH2‐ASNFD), compared to the average length of 10 residues for chemokine ligands. We have investigated how well CCL20 tolerates manipulation of the N‐terminus by monitoring binding affinity of variants and their ability to activate the receptor. We show the CCL20 N‐terminus tolerates truncation of up to 3 residues, extension by up to 5 additional residues, and point mutations at 4 of 5 positions with minimal loss of binding affinity and minimal impairment in ability to stimulate calcium mobilization, inositol triphosphate accumulation, chemotaxis, and β‐arrestin‐2 recruitment. Mutation of the fifth residue, aspartate, to alanine or lysine has a dramatic impact on binding affinity for CCR6 and ligand potency. We postulate CCL20 does not activate CCR6 through the canonical two‐step/two‐site mechanism of CKR activation.