The heterodimerization of platelet-derived chemokines James Carlson a , Sarah A. Baxter a , Didier Dréau b, c , Irina V. Nesmelova b, d, ⁎ a Analytical Sciences Laboratory, David H. Murdock Research Institute, 150 Research Campus Dr., Kannapolis, NC 28081, USA b Center for Biomedical Engineering and Science, University of North Carolina, 9201 University City Blvd., Charlotte, NC 28223, USA c Department of Biology, University of North Carolina, 9201 University City Blvd., Charlotte, NC 28223, USA d Department of Physics and Optical Science, University of North Carolina, 9201 University City Blvd., Charlotte, NC 28223, USA abstract article info Article history: Received 11 July 2012 Received in revised form 14 September 2012 Accepted 16 September 2012 Available online 23 September 2012 Keywords: Chemokines Heterodimer Heterophilic interactions Mass spectrometry (MS) Co-immunoprecipitation Platelet Chemokines encompass a large family of proteins that act as chemoattractants and are involved in many bio- logical processes. In particular, chemokines guide the migration of leukocytes during normal and inflamma- tory conditions. Recent studies reveal that the heterophilic interactions between chemokines significantly affect their biological activity, possibly representing a novel regulatory mechanism of the chemokine activi- ties. The co-localization of platelet-derived chemokines in vivo allows them to interact. Here, we used nano-spray ionization mass spectrometry to screen eleven different CXC and CC platelet-derived chemokines for possible interactions with the two most abundant chemokines present in platelets, CXCL4 and CXCL7. Re- sults indicate that many screened chemokines, although not all of them, form heterodimers with CXCL4 and/ or CXCL7. In particular, a strong heterodimerization was observed between CXCL12 and CXCL4 or CXCL7. Compared to other chemokines, the main structural difference of CXCL12 is in the orientation and packing of the C-terminal alpha-helix in relation to the beta-sheet. The analysis of one possible structure of the CXCL4/CXCL12 heterodimer, CXC-type structure, using molecular dynamics (MD) trajectory reveals that CXCL4 may undergo a conformational transition to alter the alpha helix orientation. In this new orientation, the alpha-helix of CXCL4 aligns in parallel with the CXCL12 alpha-helix, an energetically more favorable con- formation. Further, we determined that CXCL4 and CXCL12 physically interact to form heterodimers by co-immunoprecipitations from human platelets. Overall, our results highlight that many platelet-derived chemokines are capable of heterophilic interactions and strongly support future studies of the biological im- pact of these interactions. © 2012 Elsevier B.V. All rights reserved. 1. Introduction Chemokines are small regulatory proteins with diverse functions. Initially they have been classified into a family based on their ability to chemoattract different subsets of leukocytes [1,2]. However, many more important biological activities of chemokines are being discovered [3–7]. All chemokines are grouped into subfamilies according to the po- sition of two N-terminal cysteine residues [8]. In the largest subfamilies, CXC and CC, cysteines are separated by a single amino acid residue or adjacent to each other. Although proteins hardly ever function in isolation, the biology of chemokines is commonly explained as if each chemokine acts independently of other chemokines. The chemokine network in humans includes nearly 50 chemokine ligands which are involved a va- riety of intermolecular interactions including homo-oligomerization, the interactions with G protein-coupled receptors (GPCRs) and glycos- aminoglycans (GAGs) [9,10]. The recently demonstrated heterophilic interactions between chemokines, i.e. the formation of heterodimers and possibly higher order hetero-oligomers, greatly increased the di- versity and the complexity of the chemokine network activities and reg- ulations [11]. From the biophysics perspective, the structural homology of the chemokine monomers [12] forms the basis for chemokine heterodimerization. Each monomer has an unstructured N-terminus followed by a beta-sheet, composed of three anti-parallel beta- strands, and a C-terminal alpha-helix. Secondary structure elements vary in length to some extent, and the orientation of the C-terminal helix relative to beta-sheet may vary. However, overall monomers of different chemokines can be superimposed within less than 3 Å for Cα atoms of secondary structure motifs. Most chemokines tend to form dimers. Consequently, structurally similar chemokine mono- mers can be mutually interchangeable in a dimer if the placement of residues at the intermonomer interface in a mixed complex is Biochimica et Biophysica Acta 1834 (2013) 158–168 Abbreviations: MD, molecular dynamics; ESI-MS, electrospray ionization mass spectrometry; co-IP, co-immunoprecipitation; Western Blot, WB; SPR, surface plasmon resonance; M r , molecular weight; NMR, nuclear magnetic resonance spectroscopy; PDB, Protein Data Bank; ACN, acetonitrile; GPCR, G protein-coupled receptor; GAG, glycosaminoglycan; RMSD, root-mean-square deviation ⁎ Corresponding author at: Grigg Hall 306, Department of Physics and Optical Sci- ences, University of North Carolina, 9201 University City Blvd., Charlotte, NC 28223, USA. Tel.: +1 704 687 8145; fax: +1 704 687 8197. E-mail address: Irina.Nesmelova@uncc.edu (I.V. Nesmelova). 1570-9639/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.bbapap.2012.09.010 Contents lists available at SciVerse ScienceDirect Biochimica et Biophysica Acta journal homepage: www.elsevier.com/locate/bbapap