Mapping Interaction Sites on Human Chemokine Receptors by Deep Mutational Scanning

Abstract

Chemokine receptors CXCR4 and CCR5 regulate WBC trafficking and are engaged by the HIV-1 envelope glycoprotein gp120 during infection. We combine a selection of human CXCR4 and CCR5 libraries comprising nearly all of ∼7000 single amino acid substitutions with deep sequencing to define sequence-activity landscapes for surface expression and ligand interactions. After consideration of sequence constraints for surface expression, known interaction sites with HIV-1-blocking Abs were appropriately identified as conserved residues following library sorting for Ab binding, validating the use of deep mutational scanning to map functional interaction sites in G protein-coupled receptors. Chemokine CXCL12 was found to interact with residues extending asymmetrically into the CXCR4 ligand-binding cavity, similar to the binding surface of CXCR4 recognized by an antagonistic viral chemokine previously observed crystallographically. CXCR4 mutations distal from the chemokine binding site were identified that enhance chemokine recognition. This included disruptive mutations in the G protein-coupling site that diminished calcium mobilization, as well as conservative mutations to a membrane-exposed site (CXCR4 residues H79 and W161) that increased ligand binding without loss of signaling. Compared with CXCR4-CXCL12 interactions, CCR5 residues conserved for gp120 (HIV-1 BaL strain) interactions map to a more expansive surface, mimicking how the cognate chemokine CCL5 makes contacts across the entire CCR5 binding cavity. Acidic substitutions in the CCR5 N terminus and extracellular loops enhanced gp120 binding. This study demonstrates how comprehensive mutational scanning can define functional interaction sites on receptors, and novel mutations that enhance receptor activities can be found simultaneously.

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

“…These included the interactions between CXCR4 E26 and CXCL12 R47, along with CXCR4 D20 and CXCL12 K56 ( Fig 5, S1 Data). Previous one-sided mutagenesis studies have suggested a role for D20 and E26 in CXCL12 binding [53,54]; however, their functional role has not been verified. Moreover, their exact pairing with CXCL12 residues in the complex has never been explored.…”

Molecular determinants of chaperone interactions on MHC-I for folding and antigen repertoire selection

“…The Ctermini of myc-tagged MHC-I were fused to VN (a.a. V1-A154 of Venus I152L mutant) (71). Cloning of tagged CXCR4 fusions to VN and VC are previously described (72). All constructs were cloned into pCEP4 (Invitrogen), and targeted mutations were generated by site directed mutagenesis and confirmed with DNA sequencing.…”

“…The media was replaced 2 hours post transfection. Under these conditions, cells typically acquire no more than a single coding sequence (72).…”

TLmutation: predicting the effects of mutations using transfer learning

“…9,31 There are more than 20 chemokine receptors in the human body, 40 of which only 2 have a complete mutational dataset. 41 Evaluating TLmutation algorithms on these two datasets will allow us to uncover essential insights into other chemokine receptors and HIV pathology.…”

“…In this case study, we utilized available single point mutation datasets for two proteins, CXCR4 and CCR5, and two different experiments, expression level and bimolecular fluorescence complementation (BiFC) assay. 41 Sequence identity and similarity between CXCR4 and CCR5 are ∼ 30% and 50%, respectively ( Figure 5). The proposed supervised and unsupervised TLmutation algorithms were implemented and shown for one case of supervised transfer and four different cases of unsupervised transfer.…”