NMR Structure of Lung Surfactant Peptide SP-B_11-25

Abstract

Surfactant protein B (SP-B) is a 79-residue essential component of lung surfactant, the film of lipid and protein lining the alveoli, and is the subject of great interest for its role in lung surfactant replacement therapies. Here we report circular dichroism results and the solution NMR structure of SP-B(11-25) (CRALIKRIQAMIPKG) dissolved in CD(3)OH at 5 degrees C. This is the first report of NMR data related to the protein SP-B, whose structure promises to help elucidate the mechanism of its function. Sequence-specific resonance assignments were made for all observable (1)H NMR signals on the basis of standard 2D NMR methods. Structures were determined by the simulated annealing method using restraints derived from 2D NOESY data. The calculations yielded 17 energy-minimized structures, three of which were subjected to 0.95 ns of restrained dynamics to assess the relevance of the static structures to more realistic dynamic behavior. Our CD and NMR data confirm that this segment is an amphiphilic alpha helix from approximately residue L14 through M21. The backbone heavy-atom RMSD for residues L14 through M21 is 0.09 +/- 0.12 A, and the backbone heavy-atom RMSD for the whole peptide is 0.96 +/- 2.45 A, the difference reflecting fraying at the termini. Aside from the disordered termini, the minimized structures represent dynamic structures well. Structural similarity to the homologous regions of related saposin-like proteins and the importance of the distribution of polar residues about the helix axis are discussed.

NMR Structures of the C-Terminal Segment of Surfactant Protein B in Detergent Micelles and Hexafluoro-2-propanol

“…Thus, the SP-B monomer can be expected to consist of a bundle of four or five helices, stabilized by three disulfide bonds, two of which link the C-terminal and N-terminal helices. The structure of segments of SP-B from near its N-terminus has previously been probed in lipid by FTIR (39) and in methanol by NMR (40). This study examines the structure of the 16 C-terminal residues of SP-B (SP-B CTERM ) which contains the predicted C-terminal helix and several residues N-terminal to the putative helix.…”

Functional importance of the NH2-terminal insertion sequence of lung surfactant protein B

“…Polarization modulation infrared reflectance absorption spectroscopy of the SP-B 1-25 peptide in 4:1 DPPC:DOPG monolayers shows a reorientation of the peptide from parallel to perpendicular with respect to the interface at high compression (59), placing the peptide in a configuration that would support extrusion of discoid bicelles. Moreover, the amphipathic helix region of SP-B, estimated to span at least 8 amino acids (residues 14 -21) (38), is ϳ12 Å in length while the lipid tail region in the bilayer spans ϳ20 Å (73), suggesting that two helices could comfortably span the length of the tail region of the bilayer as outlined in Fig. 7C.…”

“…While there are no high-resolution crystal or NMR structures of the native SP-B protein, the structure of the NH 2 terminus up to residue 25 is well defined as determined by a combination of NMR, FTIR, and Raman spectroscopy (6,22,38,53,59,71). Residues 1-9 (FPIPLPYCW), proposed to act as an "insertion sequence," comprise a hydrophobic region containing a poly-proline-like sequence.…”

“…This region also contains a tryptophan residue at position 9 thought to help anchor the peptide at the fluid/lipid interface (12,72). The remainder of the sequence, residues 10 -25 (LCRALIKRIQAMPIAMIPKG), is an amphipathic helix with the hydrophilic side lined with four cationic residues and the hydrophobic side dominated by alanine, leucine, and isoleucine residues (38). The functional effect of structural motifs found in SP-B 1-25 has been tested by the synthesis of peptoids based on this general design with a hydrophobic, helical insertion region with aromatic side chains, which show comparable biomimetic surface activity to SP-B 1-25 (58).…”

Surfactant protein B: unambiguously necessary for adult pulmonary function

“…Although similar in hydrophobicity and surface activity to surfactant protein C (29,30), surfactant protein B probably arose considerably earlier in evolution than surfactant protein C. Unlike the hydrophilic surfactant proteins A (also detectable early in pulmonary evolution) and D, surfactant protein B is surface active: it contains 5 amphiphilic ␣-helices that interact with the surfactant lipid monolayer to reduce breakup of anionic lipid headgroups during the folding transition of the pulmonary surfactant's phospholipid monolayer and loss of these same headgroups to the subphase upon monolayer collapse (10,19). Surfactant protein B also lowers surface tension by disrupting attractive forces between water molecules (19). The evolutionary conservation of surfactant protein B and its surface tension-lowering characteristics in air-breathing mammals both predict a critical role in adult lung function.…”