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The structure of latherin, a surfactant allergen protein from horse sweat and saliva.

Vance SJ, McDonald RE, Cooper A, Smith BO, Kennedy MW - J R Soc Interface (2013)

Bottom Line: Its surfactant activity is intrinsic to the protein in its native form, and is manifest without associated lipids or glycosylation.Intrinsically surface-active proteins are relatively rare in nature, and this is the first structure of such a protein from mammals to be reported.Both its conformation and proposed method of action are different from other, non-mammalian surfactant proteins investigated so far.

View Article: PubMed Central - PubMed

Affiliation: School of Chemistry, University of Glasgow, Glasgow G12 8QQ, UK.

ABSTRACT
Latherin is a highly surface-active allergen protein found in the sweat and saliva of horses and other equids. Its surfactant activity is intrinsic to the protein in its native form, and is manifest without associated lipids or glycosylation. Latherin probably functions as a wetting agent in evaporative cooling in horses, but it may also assist in mastication of fibrous food as well as inhibition of microbial biofilms. It is a member of the PLUNC family of proteins abundant in the oral cavity and saliva of mammals, one of which has also been shown to be a surfactant and capable of disrupting microbial biofilms. How these proteins work as surfactants while remaining soluble and cell membrane-compatible is not known. Nor have their structures previously been reported. We have used protein nuclear magnetic resonance spectroscopy to determine the conformation and dynamics of latherin in aqueous solution. The protein is a monomer in solution with a slightly curved cylindrical structure exhibiting a 'super-roll' motif comprising a four-stranded anti-parallel β-sheet and two opposing α-helices which twist along the long axis of the cylinder. One end of the molecule has prominent, flexible loops that contain a number of apolar amino acid side chains. This, together with previous biophysical observations, leads us to a plausible mechanism for surfactant activity in which the molecule is first localized to the non-polar interface via these loops, and then unfolds and flattens to expose its hydrophobic interior to the air or non-polar surface. Intrinsically surface-active proteins are relatively rare in nature, and this is the first structure of such a protein from mammals to be reported. Both its conformation and proposed method of action are different from other, non-mammalian surfactant proteins investigated so far.

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Latherin unfolding at an air : water interface. Speculative model of how latherin may transform from its fold in the bulk phase to an opened-out conformation at an air : water interface, thereby exposing its apolar interior to the air. The model shows three stages, from left to right: latherin in the bulk phase in which recognition of the interface occurs via the relatively hydrophobic loops; initial unzipping of the two α-helices initiated from the ‘loop’ end and a final open, planar, conformation, retaining secondary structure but with the hydrophobic core exposed at the interface. There will likely be dynamic exchange between the three conformations. A similar process may apply for latherin associating with a hydrophobic solid surface.
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RSIF20130453F5: Latherin unfolding at an air : water interface. Speculative model of how latherin may transform from its fold in the bulk phase to an opened-out conformation at an air : water interface, thereby exposing its apolar interior to the air. The model shows three stages, from left to right: latherin in the bulk phase in which recognition of the interface occurs via the relatively hydrophobic loops; initial unzipping of the two α-helices initiated from the ‘loop’ end and a final open, planar, conformation, retaining secondary structure but with the hydrophobic core exposed at the interface. There will likely be dynamic exchange between the three conformations. A similar process may apply for latherin associating with a hydrophobic solid surface.

Mentions: As we have observed elsewhere [6], the structure of a surfactant protein in bulk solution does not necessarily reflect its disposition at the air : water interface. Indeed, for monomeric proteins in solution, conformational change at the interface would seem to be a requirement in order to reconcile the need for good aqueous solubility in the bulk phase, while presenting a more amphipathic appearance at an interface. Such a radical conformational change also seems to be required for latherin. Our previous neutron reflection data indicate that latherin forms a relatively thin (mono)layer approximately 10 Å deep at the air : water surface, and with an area of approximately 4350 Å2 per molecule [3]. Interestingly, neutron reflection studies show that non-specific irreversible interfacial layers sometimes observed with other proteins are much thicker in comparison, typically of order 30 Å [62–65]. The latherin cylinder in the bulk phase is about 75 Å long by about 25 Å in diameter, which is incompatible with a 10 Å layer in its fold in the bulk phase. Complete unfolding and flattening of a cylinder of these dimensions yields an area of approximately 5890 Å2, which, while accepting the crudity of this approximation, is compatible with the value obtained from neutron reflection. How latherin initially associates with an interface, and the events that follow, remain unknown, but its structure and dynamics provide both clues and topological constraints. The dynamic, unstructured, apolar side chain-rich loops are the most likely place where the protein could associate, penetrate and anchor to a surface, and the loops would have sufficient flexibility to then splay out with apolar side chains oriented towards the air or a non-polar solid substrate. Subsequent unzippering of the protein cylinder is unlikely to occur between any of the β strands because of the cooperative hydrogen bonding between them. This constraint is reinforced by the hydrogen–deuterium exchange data that identifies the inter-strand H-bonds (in particular, between strands 2 and 3) as the most stable in the molecule. Unstitching between strand 4 and helix B is unlikely given the disulfide bond that connects them approximately midway down their lengths. Assuming minimal change in the secondary structure elements, this leaves the seams between the two helices, or between helix A and strand 1 as likely fault lines. Given that the solvent-excluded interfacial area buried between the two helices is approximately 2000 Å2, and that between helix A and strand 1 is approximately 1300 Å2, the latter case appears to be the more favourable. Conversely, assuming that unfolding initiates from the apolar loops, as proposed above, the ability of helix A and strand 1 to reorient in an independent manner is likely to be inhibited by the loop that connects the two features at this end of the protein. By contrast, there is no such topological constraint on the relative orientation of the two helices. A possible unfolding sequence involving an opening between the helices is illustrated in figure 5.Figure 5.


The structure of latherin, a surfactant allergen protein from horse sweat and saliva.

Vance SJ, McDonald RE, Cooper A, Smith BO, Kennedy MW - J R Soc Interface (2013)

Latherin unfolding at an air : water interface. Speculative model of how latherin may transform from its fold in the bulk phase to an opened-out conformation at an air : water interface, thereby exposing its apolar interior to the air. The model shows three stages, from left to right: latherin in the bulk phase in which recognition of the interface occurs via the relatively hydrophobic loops; initial unzipping of the two α-helices initiated from the ‘loop’ end and a final open, planar, conformation, retaining secondary structure but with the hydrophobic core exposed at the interface. There will likely be dynamic exchange between the three conformations. A similar process may apply for latherin associating with a hydrophobic solid surface.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4043175&req=5

RSIF20130453F5: Latherin unfolding at an air : water interface. Speculative model of how latherin may transform from its fold in the bulk phase to an opened-out conformation at an air : water interface, thereby exposing its apolar interior to the air. The model shows three stages, from left to right: latherin in the bulk phase in which recognition of the interface occurs via the relatively hydrophobic loops; initial unzipping of the two α-helices initiated from the ‘loop’ end and a final open, planar, conformation, retaining secondary structure but with the hydrophobic core exposed at the interface. There will likely be dynamic exchange between the three conformations. A similar process may apply for latherin associating with a hydrophobic solid surface.
Mentions: As we have observed elsewhere [6], the structure of a surfactant protein in bulk solution does not necessarily reflect its disposition at the air : water interface. Indeed, for monomeric proteins in solution, conformational change at the interface would seem to be a requirement in order to reconcile the need for good aqueous solubility in the bulk phase, while presenting a more amphipathic appearance at an interface. Such a radical conformational change also seems to be required for latherin. Our previous neutron reflection data indicate that latherin forms a relatively thin (mono)layer approximately 10 Å deep at the air : water surface, and with an area of approximately 4350 Å2 per molecule [3]. Interestingly, neutron reflection studies show that non-specific irreversible interfacial layers sometimes observed with other proteins are much thicker in comparison, typically of order 30 Å [62–65]. The latherin cylinder in the bulk phase is about 75 Å long by about 25 Å in diameter, which is incompatible with a 10 Å layer in its fold in the bulk phase. Complete unfolding and flattening of a cylinder of these dimensions yields an area of approximately 5890 Å2, which, while accepting the crudity of this approximation, is compatible with the value obtained from neutron reflection. How latherin initially associates with an interface, and the events that follow, remain unknown, but its structure and dynamics provide both clues and topological constraints. The dynamic, unstructured, apolar side chain-rich loops are the most likely place where the protein could associate, penetrate and anchor to a surface, and the loops would have sufficient flexibility to then splay out with apolar side chains oriented towards the air or a non-polar solid substrate. Subsequent unzippering of the protein cylinder is unlikely to occur between any of the β strands because of the cooperative hydrogen bonding between them. This constraint is reinforced by the hydrogen–deuterium exchange data that identifies the inter-strand H-bonds (in particular, between strands 2 and 3) as the most stable in the molecule. Unstitching between strand 4 and helix B is unlikely given the disulfide bond that connects them approximately midway down their lengths. Assuming minimal change in the secondary structure elements, this leaves the seams between the two helices, or between helix A and strand 1 as likely fault lines. Given that the solvent-excluded interfacial area buried between the two helices is approximately 2000 Å2, and that between helix A and strand 1 is approximately 1300 Å2, the latter case appears to be the more favourable. Conversely, assuming that unfolding initiates from the apolar loops, as proposed above, the ability of helix A and strand 1 to reorient in an independent manner is likely to be inhibited by the loop that connects the two features at this end of the protein. By contrast, there is no such topological constraint on the relative orientation of the two helices. A possible unfolding sequence involving an opening between the helices is illustrated in figure 5.Figure 5.

Bottom Line: Its surfactant activity is intrinsic to the protein in its native form, and is manifest without associated lipids or glycosylation.Intrinsically surface-active proteins are relatively rare in nature, and this is the first structure of such a protein from mammals to be reported.Both its conformation and proposed method of action are different from other, non-mammalian surfactant proteins investigated so far.

View Article: PubMed Central - PubMed

Affiliation: School of Chemistry, University of Glasgow, Glasgow G12 8QQ, UK.

ABSTRACT
Latherin is a highly surface-active allergen protein found in the sweat and saliva of horses and other equids. Its surfactant activity is intrinsic to the protein in its native form, and is manifest without associated lipids or glycosylation. Latherin probably functions as a wetting agent in evaporative cooling in horses, but it may also assist in mastication of fibrous food as well as inhibition of microbial biofilms. It is a member of the PLUNC family of proteins abundant in the oral cavity and saliva of mammals, one of which has also been shown to be a surfactant and capable of disrupting microbial biofilms. How these proteins work as surfactants while remaining soluble and cell membrane-compatible is not known. Nor have their structures previously been reported. We have used protein nuclear magnetic resonance spectroscopy to determine the conformation and dynamics of latherin in aqueous solution. The protein is a monomer in solution with a slightly curved cylindrical structure exhibiting a 'super-roll' motif comprising a four-stranded anti-parallel β-sheet and two opposing α-helices which twist along the long axis of the cylinder. One end of the molecule has prominent, flexible loops that contain a number of apolar amino acid side chains. This, together with previous biophysical observations, leads us to a plausible mechanism for surfactant activity in which the molecule is first localized to the non-polar interface via these loops, and then unfolds and flattens to expose its hydrophobic interior to the air or non-polar surface. Intrinsically surface-active proteins are relatively rare in nature, and this is the first structure of such a protein from mammals to be reported. Both its conformation and proposed method of action are different from other, non-mammalian surfactant proteins investigated so far.

Show MeSH
Related in: MedlinePlus