Structural and biochemical analyses reveal a monomeric state of the bacterial lipocalin Blc.
Acta Crystallogr D Biol Crystallogr
; 66(Pt 12): 1308-15, 2010 Dec.
Article
en En
| MEDLINE
| ID: mdl-21123871
ABSTRACT
The first bacterial member of the lipocalin protein family, Blc, was identified in Escherichia coli as an outer membrane lipoprotein that is expressed under conditions of environmental stress. Previous crystallographic studies in space group P212121 with two molecules per asymmetric unit, supported by static light-scattering experiments in solution, indicated that Blc may form a functional homodimer with lysophospholipid binding activity. Here, a new crystal structure of recombinant Blc in space group I4122 with one molecule in the asymmetric unit is described. The crystal packing differs considerably from that observed previously, which was determined using an N-terminally extended version of Blc dubbed `Blc-X'. In particular, the characteristic large interface region that was previously described as being responsible for stable dimer formation is absent in the I4122 crystal structure. Thus, the dimerization behaviour of Blc-X was most likely to be caused by the additional N-terminal peptide segment resulting from the cloning strategy employed. In contrast, we used a native-like N-terminus for Blc with just the lipid-anchored first Cys residue replaced by Ala. The fully monomeric status of this recombinant version of Blc in solution was confirmed by size-exclusion chromatography as well as analytical ultracentrifugation. Consequently, these data shed new light on the previously postulated lipid-binding mechanism and biological role of Blc. Beyond this, our findings illustrate that cloning artefacts, which frequently result from recombinant protein production for structural studies, must be considered with special caution when interpreting oligomerization and/or conformational effects.
Texto completo:
1
Colección:
01-internacional
Base de datos:
MEDLINE
Contexto en salud:
3_ND
Problema de salud:
3_neglected_diseases
/
3_zoonosis
Asunto principal:
Proteínas de la Membrana Bacteriana Externa
/
Proteínas de Escherichia coli
/
Escherichia coli
/
Lipocalinas
Tipo de estudio:
Prognostic_studies
Límite:
Humans
Idioma:
En
Revista:
Acta Crystallogr D Biol Crystallogr
Año:
2010
Tipo del documento:
Article
País de afiliación:
Alemania