Functional anatomy of the full length CXCR4-CXCL12 complex systematically dissected by quantitative model-guided mutagenesis

Abstract

One sentence summary: A systematic structure-guided mutagenesis study of chemokine receptor CXCR4 reveals novel insights into epitopes regulating ligand recognition, ligand specificity and CXCL12-mediated signaling.Abstract: Due to their prominent role in development and infamy in cancer and HIV, the chemokine receptor CXCR4 and its ligand, CXCL12, have been the subject of numerous structural and functional studies. Nevertheless, a high resolution structure of the CXCR4-CXCL12 complex has not been reported. Even with several alternative computational models of the complex at hand, the relative contributions of different interaction epitopes to ligand binding, ligand selectivity and signaling are not readily apparent. Here, building upon our latest structural model, we employed a systematic mutagenesis strategy to dissect the functional anatomy of the of CXCR4-CXCL12 complex. Key charge swap mutagenesis experiments supported pairwise interactions between oppositely charged residues in the receptor and chemokine, confirming the accuracy of the predicted orientation of the chemokine relative to the receptor, while also providing insight into ligand selectivity. Progressive deletion of N-terminal residues revealed an unexpected contribution of the receptor N-terminus to chemokine signaling; this finding challenges a longstanding "two-site" hypothesis about the essential features of the receptor-chemokine interaction where the N-terminus is purported to only contribute to binding affinity. The results suggest that while the interaction of the chemokine N-terminus with the receptor binding pocket is the key driver of signaling, the signaling amplitude depends on the extent to which the receptor N-terminus binds the chemokine. Along with systematic characterization of other epitopes, the current data allow us to propose a comprehensive experimentally-consistent structural model for how the chemokine binds CXCR4 and initiates signal transmission through the receptor TM domain. IntroductionChemokine receptors are members of the Class A family G protein-coupled receptors (GPCRs) that are best known for their role in controlling the migration of cells, particularly leukocytes in the context of immune system function. They are activated by chemokines, small 8-10 kDa secreted proteins, via a mechanism that has long been described as involving "two sites" or "two steps" (1-5). According to the two-site mechanism, the globular domain of the chemokine binds to the N-terminus of a receptor (referred to as chemokine recognition site 1, CRS1) and contributes primarily to the stability of the complex whereas the N-terminus of the chemokine binds in the transmembrane (TM) domain binding pocket of the receptor (chemokine recognition site 2, CRS2) to activate signaling (6). The distinction between these two sites arose from the general observation that mutations in chemokine N-termini produce a disproportionately large effect on receptor signaling efficacy compared to mutations in the chemokine globular domains (7,8), with correspondin...

Crosslinking-guided geometry of a complete CXC receptor-chemokine complex and the basis of chemokine subfamily selectivity

“…Finally, the selectivity insights from the model extend beyond CRS0.5: we also propose the basis for unique engagement of the proximal chemokine N-terminus in the major subpocket of the receptor, where the conserved arginine residue preceding the chemokine CXC motif (R8 in CXCL12) is coordinated by the conserved acid in the receptor TM6 position 6.58 (D262 6.58 in CXCR4, Fig 4G). This engagement depends on a unique CXC-motif-dependent "bend" [6] in the chemokine N-terminus [6,60], and may be a distinguishing feature of CXC complexes (S10 Fig). In the case of CXCR4-CXCL12, it is consistent with the reports of deleterious effects of D262 6.58 and R8 mutations, respectively [22,25,33]. It is also consistent with earlier observations of the critical role of the "ELR motif" in other, neutrophil-activating CXC chemokines [66,67], as well as the corresponding TM6 residue of neutrophil chemotactic CXC receptors [68,69] (D265 6.58 in CXCR1).…”

“…Building upon prior experimental and modeling efforts that focused on CRS2 [6,33], we refined the model and completed it by revealing the structural basis for the binding of the full receptor N-terminus to various regions of the chemokine globular core. Consistent with pharmacological evidence of the involvement of receptor distal N-terminus in chemokine recognition [18,22], we elucidated the geometry of its interactions with the chemokine β1-strand, and validated the so-called CRS0.5 previously proposed for a homologous receptor [34]. The complete model provides insights into the role of select polar residues in the receptor and the chemokine, and explains the effects of receptor tyrosine sulfation.…”

“…Many GPCRs have extended flexible N-termini, which are often post-translationally modified and contribute to ligand binding and receptor signaling [55]. For chemokine receptors, the N-terminus is known to be a determinant of chemokine affinity and to contribute to specificity [14,17]}; it is also increasingly being appreciated for its role in signaling efficacy [18,22,23]. However, because the distal N-termini of receptors are invariably unresolved in receptor-chemokine crystal structures, the structural basis for their regulation of chemokine affinity and signaling remains elusive.…”

“…As a result, we not only successfully recapitulated the CRS1 consistent with earlier NMR studies, models and crystal structures of homologous receptors, but also established the geometry of the novel CRS0.5, where the distal N-terminus of CXCR4 extends into an anti-parallel β-sheet with the β1-strand of CXCL12. The crosslinking-derived proximities were combined with molecular modeling to produce a complete atomic-resolution model of the complex between CXCL12 and full-length CXCR4, which was then validated prospectively both through loss-of-function and reciprocal charge reversal mutagenesis (the latter an orthogonal experimental technique for testing pairwise residue interactions (S1 Table and [22])).…”

“…For many receptorchemokine complexes, the N-terminus is known to play an important role in affinity and selectivity [15][16][17][18], both of which may be further fine-tuned by post-translational sulfation of tyrosine residues [19,20]. Furthermore, the distal N-terminus has been proposed to be a determinant of chemokine-mediated signaling [18,21,22], and to contribute to signaling bias [23]. These studies established the functional importance of receptor N-termini; however, their interaction geometry with the chemokines remains elusive (S1 Table), which is a critical barrier for our understanding of how chemokines control diverse signaling events.…”