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Structural landscape of the Chemokine Receptor system

Chemokine receptors (CKRs) belong to a subfamily of G-protein-coupled receptors (GPCRs) and play a crucial role in inflammation and immune responses. CKRs can be classified into typical CKRs, atypical CKRs (ACKRs) which lack G-protein signaling, and viral CKRs. The chemokine system exhibits great versatility, with more than 50 chemokines interacting with over 20 receptors expressed on various cell types.

Thanks to advancements in cryo-electron microscopy (cryo-EM) technology, there has been a rapid increase in the number of experimental structures depicting chemokine receptor-chemokine complexes and revealing the diverse and multifaceted nature of the chemokine receptor system. Currently, there are more than 40 available structures of chemokines and their receptors in the Protein Data Bank (PDB) which reveal common structural features, such as the presence of seven transmembrane domains (TMs 1-7), a bridge between TM7 and the N-terminus (Szpakowska, Bercoff et al. 2014) and a TxP motif in TM2 (Govaerts, Blanpain et al. 2001).

A recent comparative analysis study of all structures of CKRs characterizes the molecular recognition processes governing the CKR system providing an overview of the chemokine binding mode and activation mechanisms (Urvas, Lauri & Kellenberger. Esther 2023).

Chemokine Binding Mode – from CRS1 to CRS3

Chemokines possess a conserved tertiary structure known as the interleukin 8-like chemokine superfamily which is characterized by a flexible N-terminus, N-loop, three anti-parallel β-strands connected by 30s- and 40s-loops, and a C-terminal α-helix. The subfamily-defining CC/CXC/CX3C/XC motif is located in the N-loop and forms two conserved disulfide bridges, which connect the N-loop to the 30s-loop and the β3-strand, thereby anchoring the distal N-terminus to the core of the chemokine. When comparing the CKRs structures complexes, the disulfide bridges formed between the N-loop and the β3-strand align well, although the position of the disulfide bridges between the N-loop and the 30s-loop varies depending on the subfamily (CC, CXC, or CX3C). The spacing between the cysteine residues in the N-loop determines the subfamily-specific arrangement of the distal N-terminus and the 30s-loop, both of which play a crucial role in the recognition of chemokine receptors. This structural variation may explain why chemokine receptors typically recognize chemokines from only one specific subfamily.

Chemokine-CKR interaction follows a common pattern which is generally described by the classic “two-site” model (Crump, Gong et al. 1997) where the chemokine N-loop and β3-strand/40s-loop bind to the receptor N-terminus (CRS1), and the N-terminus assumes diverse binding modes in the 7TM cavity of the receptor (CRS2). These two sites are observed in all typical CKR-chemokine structures. Additionally, other conserved binding features, including CRS1.5 and CRS3, have been identified. CRS1.5 is an intermediate recognition site between CRS1 and CRS2, first described in the CXCR4-vCCL2 structure (Qin, Kufareva et al. 2015), where a proline-cysteine (PC) motif in the receptor N-terminus interacts closely with the N-loop-β3-strand disulfide bridge of the chemokine promoting the chemokine N-terminus orientation towards CRS2 and the receptor N-terminus toward CRS1. The 30s-loop of chemokines can bind either on top of the extracellular loop 2 (ECL2) or inside the major binding pocket, known as CRS3 which is characterized by three different binding modes: CC chemokines with deep binding, CC/CXC chemokines with shallow binding near TM5, and CX3C chemokines with shallow binding near the ECL2 hairpin.

CKRs are characterized by a seven transmembrane (7TM) fold structure consisting of three intracellular loops (ICLs 1-3) and three extracellular loops (ECLs 1-3). The N-terminal domains of CKRs has multiple sites for post-translational modifications, including tyrosine sulfation and O-glycosylation (Szpakowska, Fievez et al. 2012, Scurci, Akondi et al. 2021, Verhallen, Lackman et al. 2023), which provide extra negative charges to the N-terminus thereby increasing binding affinity and potency at CRS1. Two conserved disulfide bridges are found in chemokine receptors - one connects the N-terminus to the extracellular tip of the seventh transmembrane helix (TM7) near ECL3, while the other links TM3 with ECL2, which is common to the broader class A GPCR family.

The arrangement of the seven transmembrane helices creates a ligand-binding cavity on the extracellular side of the receptor.

Activation in Class A GPCRs

Ligand binding in the orthosteric site leads to diverse activation mechanisms that converge at the transducer coupling region, resulting in the opening of the intracellular binding site. This involves an outward swing of the intracellular transmembrane helix 6 (TM6) coupled with an inward movement of TM7. Conformational changes between inactive and active states are facilitated by a "microswitch network" composed of conserved sequence motifs which include the W6.48xP6.50 motif and the P5.50I3.40F6.44 motif which are located below the 7TM cavity in the middle of the transmembrane region, and the N7.49P7.50xxY7.53 and D3.49R3.50Y3.51 motifs, located near the intracellular ends of TM7 and TM3, respectively (Weis and Kobilka 2018). All CKRs studied show the canonical active conformation, except CX3CR1 which displays a significantly smaller outward tilt of TM6, and instead TM7 and the intracellular helix 8 move outward from the receptor core to make room for the G-protein (Lu, Zhao et al. 2022). As a result of the unique active conformation, the residues in the microswitch network show non-canonical configurations, indicating the versatility of the GPCR architecture for different activation mechanisms.

The road map of CCR5 and CCR2 activation

The structure of CCR5 has been studied in complex with various ligands, including endogenous chemokine agonists (CCL5 and CCL3), a chemokine super-agonist ([6P4]CCL5), a chemokine antagonist ([5P7]CCL5), and a small-molecule inverse agonist (maraviroc) (Tan, Zhu et al. 2013, Zheng, Han et al. 2017, Isaikina, Tsai et al. 2021, Zhang, Chen et al. 2021). These structures provide a comprehensive understanding of the mechanisms of agonism and antagonism in chemokine receptor activation. The agonist ligands (CCL5, CCL3, and [6P4]CCL5) bind to the 7TM cavity with their N-termini adopting a hook-like conformation, whereas the chemokine antagonist ([5P7]CCL5) folds into a helical shape due to bulky hydrophobic residues in its proximal N-terminus, resulting in a shallower binding. Common to all four chemokines, the distal N-termini interact with a hydrophobic surface at the bottom of the binding site, while polar interactions formed with specific residues.

By examining both the active and inactive states of CCR5, it becomes evident that there are four distinct pathways through which structural alterations in the binding site influence Y2446.44 of the P5.50I3.40Y6.44 motif ultimately driving receptor activation: a) Route 1 - hydrogen bonding to Y2516.51 by CCL3 or CCL5; b) Route 2 - characterized by hydrogen bonding to E2837.39 by A4 of CCL3; c) Route 3 - characterized by steric effects between TM2 and Y3 of CCL5; d) Route 4 - characterized by a deep binding of P3 of [6P4]CCL5. The active state structure of CCR2 with its endogenous agonist CCL2, as well as inactive states with two different small-molecule antagonists have been successfully determined. Comparison between the active and inactive structures reveal conformational shifts that mirror the activation routes 1 and 2 observed in CCR5.

Biased Agonism – CCR1 as a model CKR

Three active-state structures of CCR1 have been characterized, all of which are bound to a G-protein - two of the structures involve complex formation with endogenous N-terminal truncation variants of CCL15, namely CCL15L (residues 26-92) and CCL15M (residues 27-92), while the third structure lacks a ligand (Shao, Shen et al. 2022). It has been demonstrated that the shorter truncation variant, CCL15L, exhibits bias toward G-protein signaling, which has been structurally related to a conformational change of Y2917.43 tilted toward TM2, a model supported by mutagenesis experiments (Shao, Shen et al. 2022). The structural analysis of CCR1 demonstrates how the interaction or absence of interaction with a specific side chain can dictate whether an agonist exhibits a bias toward G-protein signaling or not, although the precise molecular signature responsible for G-protein activation by CCR1 remains unknown, primarily due to the absence a structure in the inactive state.

The Non-canonical Toggle Switch 6.48 - CCR6 as a model CKR

The structure of CCR6 bound to CCL20 reveals two significant features at CRS2: a shallow binding mode within the transmembrane binding site and limited interaction with the 7TM bundle. One hypothesis regarding CCR6 activation by CCL20 suggests that the crucial binding feature is the E19845.51–NH2 salt bridge (Nelson, Boyd et al. 2001), with molecular dynamics simulations indicating that the stability of the salt bridge depends on the position of NH2, which is most stable at position 1 (Wasilko, Johnson et al. 2020). The absence of an inactive-state structure of CCR6 makes it challenging to confirm the activation mechanism. However, comparing CCR6 with closely related receptors, CCR7 and CCR9, in their inactive states offers some insights: outward movements of TM3, TM4, and TM6 and an inward movement of TM5 are observed, supporting the hypothesis that CCL20 adjusts the position of TM7 relative to ECL2. These receptors share a glutamine residue at the "toggle switch" position 6.48 and the disruption of a hydrogen-bond network surrounding this non-canonical toggle switch results in the separation of TM3 and TM6 following chemokine binding to the upper part of CRS2.

Constitutive Activity of the viral CKR US28 and “Chemokine Scavenger” ACKR3

Both US28 and ACKR3 are constitutively active receptors, meaning they can signal independently of agonist (Casarosa, Bakker et al. 2001, Luker, Steele et al. 2010). US28 evolved high constitutive activity to evade host immunity, whereas ACKR3 functions as a homeostatic chemokine "scavenger" by downregulating extracellular chemokine gradients through constitutive internalization and recycling of chemokines without activating G-protein pathways (Randolph-Habecker, Rahill et al. 2002, Luker, Steele et al. 2010, Tsutsumi, Maeda et al. 2022). Experimental 3D structures of both receptors reveal that they employ distinct mechanisms for constitutive activity.

In class A GPCRs, the inactive conformation is stabilized by the inactive configuration of the D3.49R3.50Y3.51motif near the intracellular end of TM3. However, in US28, the factors stabilizing the inactive state of this motif are absent. In addition, it has a glutamate residue at position 3.45, which is not found in human class A GPCRs, and it does not have an acidic residue at position 6.30. These factors make the active state more favorable in US28. In ACKR3, the presence of a tyrosine at position 6.40 plays a role in its constitutive activity (Yen, Schafer et al. 2022) which is also supported by mutagenesis studies (Han, Tachado et al. 2012, Yen, Schafer et al. 2022). However, the role of chemokine ligands in activating US28 and ACKR3 cannot be confirmed without inactive-state structures of these receptors.

Recent advancements in cryo-EM technology have led to a rapid increase in the number of experimental structures of chemokine receptors, which provide insights into the complexity of the chemokine system, involving ligand promiscuity within subfamilies, functionally selective biased ligands, and constitutively active and intrinsically biased receptors. As more experimental structures of CKRs continue to emerge, it is expected that a more detailed understanding of chemokine binding and receptor activation will pave the way for significant advancements in the development of therapeutics for a wide range of diseases.

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