Acidic pH triggers conformational changes at the NH2-terminal propeptide of the precursor of pulmonary surfactant protein B to form a coiled coil structure.
Pulmonary surfactant protein SP-B is synthesized as a larger precursor, proSP-B. We report that a recombinant form of human SP-BN forms a coiled coil structure at acidic pH. The protonation of a residue with pK=4.8±0.06 is the responsible of conformational changes detected by circular dichroism and intrinsic fluorescence emission. Sedimentation velocity analysis showed protein oligomerisation at any pH condition, with an enrichment of the species compatible with a tetramer at acidic pH. Low 2,2,2,-trifluoroethanol concentration promoted β-sheet structures in SP-BN, which bind Thioflavin T, at acidic pH, whereas it promoted coiled coil structures at neutral pH. The amino acid stretch predicted to form β-sheet parallel association in SP-BN overlaps with the sequence predicted by several programs to form coiled coil structure. A synthetic peptide ((60)W-E(85)) designed from the sequence of the amino acid stretch of SP-BN predicted to form coiled coil structure showed random coil conformation at neutral pH but concentration-dependent helical structure at acidic pH. Sedimentation velocity analysis of the peptide indicated monomeric state at neutral pH (s20, w=0.55S; Mr~3kDa) and peptide association (s20, w=1.735S; Mr=~14kDa) at acidic pH, with sedimentation equilibrium fitting to a Monomer-Nmer-Mmer model with N=6 and M=4 (Mr=14692Da). We propose that protein oligomerisation through coiled-coil motifs could then be a general feature in the assembly of functional units in saposin-like proteins in general and in the organization of SP-B in a functional surfactant, in particular.
Departamento de Bioquímica y Biología Molecular I, Facultad de Biología, Universidad Complutense, Ciudad Universitaria, 28040 Madrid, Spain.
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Bañares-Hidalgo
Departamento de Bioquímica y Biología Molecular I, Facultad de Biología, Universidad Complutense, Ciudad Universitaria, 28040 Madrid, Spain.
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Pérez-Gil
Departamento de Bioquímica y Biología Molecular I, Facultad de Biología, Universidad Complutense, Ciudad Universitaria, 28040 Madrid, Spain. Electronic address: mpestrada@bio.ucm.es.
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Estrada
D000595:Amino Acid Sequence; D002942:Circular Dichroism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry*; D011489:Protein Denaturation; D017433:Protein Structure, Secondary; D037701:Pulmonary Surfactant-Associated Protein B / Q000737:chemistry*; D011994:Recombinant Proteins / Q000737:chemistry; D016415:Sequence Alignment
D010455:Peptides; D037701:Pulmonary Surfactant-Associated Protein B; D011994:Recombinant Proteins
Coiled coil; N-terminal propeptide; Pulmonary surfactant; SP-B