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 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.
Grygiel
Tami L R
TL
Centocor Research and Development, Inc., Radnor, PA 19087, USA. grygieltlr@yahoo.com
Teplyakov
Alexey
A
Obmolova
Galina
G
Stowell
Nicole
N
Holland
Reannon
R
Nemeth
Jennifer F
JF
Pomerantz
Steven C
SC
Kruszynski
Marian
M
Gilliland
Gary L
GL
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
C111835:CCL2 protein, human; D018932:Chemokine CCL2