The G protein-coupled chemokine receptor, CXCR4, and the atypical chemokine receptor, ACKR3, play critical roles in cell migration during immune responses and organ development, through coordinated responses to a shared ligand, CXCL12. Both receptors contribute to numerous inflammatory and autoimmune diseases and are under active investigation as therapeutic targets. Nevertheless, there is currently only one FDA-approved CXCR4 antagonist (AMD3100/Plerixafor), and its use is limited to mobilizing hematopoietic stem cells for bone marrow transplants, because many of its properties are suboptimal. Therapeutic targeting of ACKR3 is at a less mature stage than CXCR4; in fact, most known compounds are agonists, and it is unclear how to antagonize this receptor. Improved compounds targeting both CXCR4 and ACKR3 are therefore needed. As an "atypical" receptor, ACKR3 is widely assumed to function only through β-arrestin (and not G proteins), and is best known for its ability to “scavenge” CXCL12 from the extracellular environment. By doing so, ACKR3 prevents downregulation of CXCR4 and maintains its responsiveness to CXCL12 gradients. When co-expressed in the same cell, ACKR3 can also alter CXCR4 signaling and trafficking via heterodimerization, sequestration of β-arrestin, and other as-yet-undeciphered mechanisms. Given that ACKR3 binds CXCL12 in an architecture similar to CXCR4, undergoes similar conformational changes upon activation, and shares all of the conserved G protein-coupling determinants, its presumed Gi incompetency is striking. Even more striking is the exceptional robustness of ACKR3 activation to ligand and receptor modifications, whereas CXCR4 activation is abrogated by the subtlest of such changes. Because of this activation-prone nature, most non-chemokine (and even small molecule) ligands activate ACKR3 association with β-arrestin, with unknown downstream consequences. Despite the role of the two receptors in disease, the structural and molecular mechanisms underlying their individual functions and their cellular crosstalk remain elusive. In this MPI proposal, the Handel and Kufareva labs combine their experimental and computational expertise, respectively, with their in-depth knowledge of chemokine receptors, to explain the distinct activation mechanisms of CXCR4 (Aim 1) and ACKR3 (Aim 2) from the standpoint of structure and dynamics, to understand how to inhibit these receptors (Aims 1 and 2), and to understand how ACKR3 regulates the function of CXCR4 (Aim 3). To achieve these aims, specific mechanistic hypotheses are probed with a combination of structural (cryo-EM and crystallography), computational (modeling and MD) and cell-based functional experiments, and complemented by unbiased discovery proteomics. These studies will deliver unprecedented insight into the function of CXCR4 and ACKR3, which will have a direct impact on the development of small molecule therapeutics and provide the rationale for blocking one or both receptor...