Research article

X-ray structure of engineered human Aortic Preferentially Expressed Protein-1 (APEG-1)

Babu A Manjasetty^1,2,8 , Frank H Niesen^4,6,7 , Christoph Scheich^4,5 , Yvette Roske^2,4 , Frank Goetz^2,4 , Joachim Behlke² , Volker Sievert^4,5 , Udo Heinemann^2,3 and Konrad Büssow^4,5

¹Protein Structure Factory, c/o BESSY GmbH, Albert-Einstein-Str. 15, 12489 Berlin, Germany

²Max-Delbrück-Centrum für Molekulare Medizin, Robert-Rössle-Str. 10, 13092 Berlin, Germany

³Institut für Chemie/Kristallographie, Freie Universität, Takustr. 6, 14195 Berlin, Germany

⁴Protein Structure Factory, Heubnerweg 6, 14059 Berlin, Germany

⁵Max-Planck-Institut für Molekulare Genetik, Ihnestr. 73, 14195 Berlin, Germany

⁶Charité Universitätsmedizin Berlin, Institut für Medizinische Physik & Biophysik, Ziegelstr. 5-9, 10098 Berlin, Germany

⁷Structural Genomics Consortium, University of Oxford, Botnar Research Centre, Oxford, OX3 7LD, UK

⁸Case Centre for Proteomics, Case Western Reserve University, Upton, New York 11973, USA

author email corresponding author email

BMC Structural Biology 2005, 5:21doi:10.1186/1472-6807-5-21

Published:	14 December 2005

Abstract

Background

Human Aortic Preferentially Expressed Protein-1 (APEG-1) is a novel specific smooth muscle differentiation marker thought to play a role in the growth and differentiation of arterial smooth muscle cells (SMCs).

Results

Good quality crystals that were suitable for X-ray crystallographic studies were obtained following the truncation of the 14 N-terminal amino acids of APEG-1, a region predicted to be disordered. The truncated protein (termed ΔAPEG-1) consists of a single immunoglobulin (Ig) like domain which includes an Arg-Gly-Asp (RGD) adhesion recognition motif. The RGD motif is crucial for the interaction of extracellular proteins and plays a role in cell adhesion. The X-ray structure of ΔAPEG-1 was determined and was refined to sub-atomic resolution (0.96 Å). This is the best resolution for an immunoglobulin domain structure so far. The structure adopts a Greek-key β-sandwich fold and belongs to the I (intermediate) set of the immunoglobulin superfamily. The residues lying between the β-sheets form a hydrophobic core. The RGD motif folds into a 3₁₀ helix that is involved in the formation of a homodimer in the crystal which is mainly stabilized by salt bridges. Analytical ultracentrifugation studies revealed a moderate dissociation constant of 20 μM at physiological ionic strength, suggesting that APEG-1 dimerisation is only transient in the cell. The binding constant is strongly dependent on ionic strength.

Conclusion

Our data suggests that the RGD motif might play a role not only in the adhesion of extracellular proteins but also in intracellular protein-protein interactions. However, it remains to be established whether the rather weak dimerisation of APEG-1 involving this motif is physiogically relevant.