Synthesis by native chemical ligation and crystal structure of human CCL2

The protein human CC chemokine ligand 2 (CCL2, also known as monocyte chemoattractant protein 1 or MCP-1) has been synthesized using a combination of solid phase peptide synthesis (SPPS) and native chemical ligation (NCL). The thioester-peptide segment was synthesized using the sulfonamide safety-catch linker and 9-fluorenylmethoxycarbonyl (Fmoc) SPPS, and pseudoproline dipeptides were used to facilitate the synthesis of both CCL2 fragments. After assembly of the full-length peptide chain by NCL, a glutathione redox buffer was used to fold and oxidize the CCL2 protein. Synthetic human CCL2 binds to and activates the CCR2 receptor on THP-1 cells, as expected. CCL2 was crystallized and the structure was determined by X-ray diffraction at 1.9-A resolution. The structure of the synthetic protein is very similar to that of a previously reported structure of recombinant human CCL2, although the crystal form is different. The functional CCL2 dimer for the crystal structure reported here is formed around a crystallographic twofold axis. The dimer interface involves residues Val9-Thr10-Cys11, which form an intersubunit antiparallel β-sheet. Comparison of the CCL2 dimers in different crystal forms indicates a significant flexibility of the quaternary structure. To our knowledge, this is one of the first crystal structures of a protein prepared using the sulfonamide safety-catch linker and NCL. © 2010 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 94: 350–359, 2010. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com