title: Synthesis by native chemical ligation and crystal structure of human CCL2.
abstract: 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 beta-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.
authors.affiliation: Centocor Research and Development, Inc., Radnor, PA 19087, USA. grygieltlr@yahoo.com
authors.forename: Tami L R
authors.initials: TL
authors.lastname: Grygiel
authors.forename: Alexey
authors.initials: A
authors.lastname: Teplyakov
authors.forename: Galina
authors.initials: G
authors.lastname: Obmolova
authors.forename: Nicole
authors.initials: N
authors.lastname: Stowell
authors.forename: Reannon
authors.initials: R
authors.lastname: Holland
authors.forename: Jennifer F
authors.initials: JF
authors.lastname: Nemeth
authors.forename: Steven C
authors.initials: SC
authors.lastname: Pomerantz
authors.forename: Marian
authors.initials: M
authors.lastname: Kruszynski
authors.forename: Gary L
authors.initials: GL
authors.lastname: Gilliland
mesh_terms: D000595:Amino Acid Sequence; D018932:Chemokine CCL2 / Q000138:chemical synthesis* / Q000737:chemistry* / Q000235:genetics; D018360:Crystallography, X-Ray; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D017510:Protein Folding; D055503:Protein Multimerization; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D011869:Radioligand Assay
chemical_list: C111835:CCL2 protein, human; D018932:Chemokine CCL2