Chemokines orchestrate leukocyte trafficking and function in health and disease. Heterophilic interactions between chemokines in a given microenvironment may amplify, inhibit, or modulate their activity; however, a systematic evaluation of the chemokine interactome has not been performed. We used immunoligand blotting and surface plasmon resonance to obtain a comprehensive map of chemokine-chemokine interactions and to confirm their specificity. Structure-function analyses revealed that chemokine activity can be enhanced by CC-type heterodimers but inhibited by CXC-type heterodimers. Functional synergism was achieved through receptor heteromerization induced by CCL5-CCL17 or receptor retention at the cell surface via auxiliary proteoglycan binding of CCL5-CXCL4. In contrast, inhibitory activity relied on conformational changes (in CXCL12), affecting receptor signaling. Obligate CC-type heterodimers showed high efficacy and potency and drove acute lung injury and atherosclerosis, processes abrogated by specific CCL5-derived peptide inhibitors or knock-in of an interaction-deficient CXCL4 variant. Atheroprotective effects of CCL17 deficiency were phenocopied by a CCL5-derived peptide disrupting CCL5-CCL17 heterodimers, whereas a CCL5 α-helix peptide mimicked inhibitory effects on CXCL12-driven platelet aggregation. Thus, formation of specific chemokine heterodimers differentially dictates functional activity and can be exploited for therapeutic targeting.

Use of oxime forming reactions has become a widely applied strategy for peptide and protein bioconjugation. The efficiency of the reaction and robust stability of the oxime product has led to the development of a growing list of methods to introduce the required ketone or aldehyde functionality site specifically into proteins. Early methods focused on site-specific oxidation of an N-terminal serine or threonine and more recently transamination methods have been developed to convert a broader set of N-terminal amino acids into a ketone or aldehyde. More recently, site-specific modification of protein has been attained through engineering enzymes involved in posttranslational modifications in order to accommodate aldehyde-containing substrates. Similarly, a growing list of unnatural amino acids can be introduced through development of selective amino-acyl tRNA synthetase/tRNA pairs combined with codon reassignment. In the case of glycoproteins, glycans can be selectively modified chemically or enzymatically to introduce aldehyde functional groups. Finally, the total chemical synthesis of proteins complements these biological and chemoenzymatic approaches. Once introduced, the oxime ligation of these aldehyde and ketone groups can be catalyzed by aniline or a variety of aniline derivatives to tune the activity, pH preference, stability and solubility of the catalyst. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.

Nuclear receptor binding to coactivator proteins is an obligate first step in the regulation of gene transcription. Nuclear receptors preferentially bind to an LXXLL peptide motif which is highly conserved throughout the 300 or so natural coactivator proteins. This knowledge has shaped current understanding of this fundamental protein-protein interaction, and continues to inspire the search for new drug therapies. However, sequence specificity beyond the LXXLL motif and the molecular functioning of flanking residues still requires urgent addressing. Here, ribosome display has been used to reassess the estrogen receptor for new and enlarged peptide recognition motifs, leading to the discovery of a potent and highly evolved PXLXXLLXXP binding consensus. Molecular modeling and X-ray crystallography studies have provided the molecular insights on the role of the flanking prolines in priming the length of the α-helix and enabling optimal interactions of the α-helix dipole and its surrounding amino acids with the surface charge clamp and the receptor activation function 2. These findings represent new structural parameters for modulating the nuclear receptor-coactivator interaction based on linear sequences of proteinogenic amino acids and for the design of chemically modified inhibitors.

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Chemical reaction rates are generally decreased at lower temperatures. Here, we report that an oxime ligation reaction in water at neutral pH is accelerated by freezing. The freezing method and its rate effect on oxime ligation are systematically studied on a peptide model system, and applied to a larger chemokine protein, containing a single acetyl butyrate group, which is conjugated to an aminooxy-labeled ligand. Our improved ligation protocol now makes it possible to efficiently introduce oxime-bond coupled ligands into proteins under aqueous conditions at low concentrations and neutral pH.