Acidic pH triggers conformational changes at the NH2-terminal propeptide of the precursor of pulmonary surfactant protein B to form a coiled coil structure

Abstract

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.

Proton Leakage Is Sensed by IM30 and Activates IM30-Triggered Membrane Fusion

“…While it remains unclear what structure is actually acquired at pH 5.5, the protein potentially contains some unconventional β-sheet structures, as, for example, described in McCubbin et al [54]. Similar CD-spectra with a shifted β-sheet minimum were, for example, observed when the structure of small peptides was monitored upon pH changes or TFE treatment [55]. The intimate environment of Trp71 also changed in a biphasic manner when the pH was lowered, indicating a structural modification involving the region containing the coiled-coil forming helices 2 and 3 ( Figure 2).…”

A model for the structure and mechanism of action of pulmonary surfactant protein B

“…Noncovalent dimers of SP-B would be formed by well-defined interactions through helix 1 and the N-terminal segments of the protein. Helix 1 of SP-B would then create a very specific dimerization surface through formation of an antiparallel coiled coil, as proposed for other proteins of the saposin family including the pro-SP-B precursor itself (39). Rings, with a diameter of ;10 nm, could then form upon association of 5 or 6 of these dimers once positioned at the surface of membranes.…”

Conformational Stability of the NH2-Terminal Propeptide of the Precursor of Pulmonary Surfactant Protein SP-B

“…When 500 mM NaCl was employed, three small transitions at 41–55°C, 51–69°C and 69–81°C could be detected and fitted to Eq (2) , with midpoint transitions of 46.7±0.2°C, 62.7±1.6°C and 75.2±0.4°C respectively. The observed changes in the propeptide secondary structure may be due to partial unfolding of protein domains or subdomains (small increases in [θ] 208 ) or to the dissociation of propeptide oligomers [ 15 ]. Alternatively, small decreases in [θ] 208 may be due to partial folding of unstructured domains existing in the connector arm of the propeptide (predictions in S2 Fig ) as observed by Uversky in other proteins [ 29 ].…”

“…This seems to indicate that the disulfide bonds formed according to the expected saposin pattern could provide a surplus of stability to the core (probably the most important part, from a functional point of view, of the protein). In addition, the acidification of the medium causes the formation of a coiled-coil structure with a stretch of the SAPB domain involved in it [ 15 ]. Moreover, the presence of salt in the buffer may be important for neutralizing the acidic residues (28%) concentrated in the 148 P-D 174 stretch of the connector arm, where the protein must hold disorder, for promoting stabilization upon oligomerization.…”

“…In this context, we have produced a recombinant form of human SP-B N , soluble and containing the microbicide module [ 13 ] and the conditions that favoured or hindered its aggregation have been studied [ 14 ]. Also we have observed that acidic pH triggers, in the propeptide, the formation of a coiled coil structure [ 15 ]. In the present work, we extend the structural dissection of the protein by analyzing the thermodynamical stability of the propeptide, and the detection of their folding intermediates, if any, in the presence of chaotropes.…”