Unique Crystal Structure of a Novel Surfactant Protein from the Foam Nest of the Frog Leptodactylus vastus

Abstract

Breeding by releasing eggs into stable biofoams (“foam nests”) is a peculiar reproduction mode within anurans, fish, and tunicates; not much is known regarding the biochemistry or molecular mechanisms involved. Lv‐ranaspumin (Lv‐RSN‐1) is the predominant protein from the foam nest of the frog Leptodactylus vastus. This protein shows natural surfactant activity, which is assumed to be crucial for stabilizing foam nests. We elucidated the amino acid sequence of Lv‐RSN‐1 by de novo sequencing with mass‐spectrometry and determined the high‐resolution X‐ray structure of the protein. It has a unique fold mainly composed of a bundle of 11 α‐helices and two small antiparallel β‐strands. Lv‐RSN‐1 has a surface rich in hydrophilic residues and a lipophilic cavity in the region of the antiparallel β‐sheet. It possesses intrinsic surface‐active properties, reducing the surface tension of water from 73 to 61 mN m−1 (15 μg mL−1). Lv‐RSN‐1 belongs to a new class of surfactants proteins for which little has been reported regarding structure or function.

The Diverse Structures and Functions of Surfactant Proteins

“…High-resolution structural analysis of Ep-Rsn-2 and Lv-Rsn-1 by NMR and X-ray crystallography, respectively, revealed no structural similarities between the proteins ( Figure 3 D,E) 12 , 15 . Lv-Rsn-1 is a 23.5-kDa protein comprising two domains [12] . The N-terminal domain comprises a bundle of six antiparallel α helices.…”

“…In the past few years, the elucidation of high-resolution 3D structures of several surfactant proteins has opened the way for more detailed structure–function analysis of this exciting class of molecules 10 , 11 , 12 , 13 , 14 , 15 , 16 , 17 , 18 . These studies reveal that the mechanisms by which surfactant proteins achieve their function are highly diverse.…”

“…Ep-RSN-2 is the major surfactant protein in the mix, with the other ranaspumins contributing to foam stability and defense against microbes. Similarly, analysis of the foam nests of an unrelated frog species, Leptodactylus vastus , revealed a mix of proteins with Lv-ranaspumin (Lv-RSN-1) being the main contributor to the interfacial activity of the mixture 12 , 53 ( Table 1 ).…”

“…It has therefore been postulated that both proteins need to undergo some conformational change to facilitate interfacial association. Two possible conformational changes have been suggested for Lv-Rsn-1: the β strands could move to increase the hydrophobic cavity or the two halves of the protein could move apart to expose the hydrophobic core [12] . For Ep-Rsn-2, clamshell-like opening of the protein in which the helix unhinges from the β sheet has been hypothesized and is consistent with neutron-scattering data [15] .…”

Systematics of the Neotropical GenusLeptodactylusFitzinger, 1826 (Anura: Leptodactylidae): Phylogeny, the Relevance of Non-molecular Evidence, and Species Accounts

“…fallax, Krintler, 1986), and the dermal glands of several species are known to produce defensive skin secretions (Cei et al, 1967), including antimicrobial peptides and other protein toxins found in L. fallax (Rollins-Smith et al, 2005;King et al, 2005a), L. labyrinthicus (Libério et al, 2014), L. laticeps (Conlon et al, 2009), L. latrans (Nascimento et al, 2004(Nascimento et al, , 2007Leite et al, 2010), L. pentadactylus (Habermehl, 1981;Barlow, 1998;King et al, 2005b;Limaverde et al, 2009;Sousa et al, 2009), L. syphax (Dourado et al, 2007). Similarly, the foam nests of L. vastus possess a novel surfactant protein (Hissa et al, 2008(Hissa et al, , 2014.…”