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Chemerin is a processed protein that acts on G protein-coupled receptors (GPCRs) for its chemotactic and adipokine activities. The biologically active chemerin (chemerin 21-157) results from proteolytic cleavage of prochemerin and uses its C-terminal peptide containing the sequence YFPGQFAFS for receptor activation. Here we report a high-resolution cryo-electron microscopy (cryo-EM) structure of human chemerin receptor 1 (CMKLR1) bound to the C-terminal nonapeptide of chemokine (C9) in complex with Gi proteins. C9 inserts its C terminus into the binding pocket and is stabilized through hydrophobic interactions involving its Y1, F2, F6, and F8, as well as polar interactions between G4, S9, and several amino acids lining the binding pocket of CMKLR1. Microsecond scale molecular dynamics simulations support a balanced force distribution across the whole ligand–receptor interface that enhances thermodynamic stability of the captured binding pose of C9. The C9 interaction with CMKLR1 is drastically different from chemokine recognition by chemokine receptors, which follow a two-site two-step model. In contrast, C9 takes an “S”-shaped pose in the binding pocket of CMKLR1 much like angiotensin II in the AT1 receptor. Our mutagenesis and functional analyses confirmed the cryo-EM structure and key residues in the binding pocket for these interactions. Our findings provide a structural basis for chemerin recognition by CMKLR1 for the established chemotactic and adipokine activities.

Machine learning-based modelling assisting functional-oriented enzyme engineering is normally built on a predefined sequence space. Efficiently defining the determinant amino acid positions upon which the combinatorial mutational library is constructed is still a challenge in protein science. Herein, we present a comprehensive investigation of modifying a recombinant DNA polymerase for efficient incorporating unnatural nucleotides \textit{via} fine-tuning of functional dynamics, including the identification of key sites/regions outside known active center, machine learning-assisted mutant screening, and the underlying mechanism of kinetics boosting. By using hundreds of training points and only dozens of machine-learning predicted samples, we found that the highly engineered enzyme’s catalytic efficiency can be further improved by at least one order of magnitude by specific mutation of two sites on finger subdomain, 485I and 451L. Contrary to the position 485 which is known to dominate local conformation of B-family DNA polymerases, position 451 is a new active site discovered by our approach. The synergy of 485I and 451L on kinetics boosting is driven by an enhanced dynamical-cooperative regulation mechanism of the state distribution of 'open/closed' conformations and the dynamics of the nucleotide of template DNA for substrate-pairing. These findings extended the boundaries of the functional dynamics engineering of complex protein enzymes and have implications in guiding the design of DNA polymerases for a broad range of biotechnological applications.

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Cisplatin is one of the most widely used anticancer drugs. Its efficiency is unfortunately severely hampered by resistance. The High Mobility Group Box (HMGB) proteins may sensitize tumor cells to cisplatin by specifically binding to platinated DNA (PtDNA) lesions. In vivo, the HMGB/PtDNA binding is regulated by multisite post-translational modifications (PTMs). The impact of PTMs on the HMGB/PtDNA complex at atomistic level is here investigated by enhanced sampling molecular simulations. The PTMs turn out to affect the structure of the complex, the mobility of several regions (including the platinated site), and the nature of the protein/PtDNA non-covalent interactions. Overall, the multisite PTMs increase significantly the apparent synchrony of all the contacts between the protein and PtDNA. Consequently, the hydrophobic anchoring of the side chain of F37 between the two cross-linked guanines at the platinated site—a key element of the complexes formation - is more stable than in the complex without PTM. These differences can account for the experimentally measured greater affinity for PtDNA of the protein isoforms with PTMs. The collective behavior of multisite PTMs, as revealed here by the synchrony of contacts, may have a general significance for the modulation of intermolecular recognitions occurring in vivo.