Structural insights into a secretory abundant heat‐soluble protein from an anhydrobiotic tardigrade,Ramazzottius varieornatus

Abstract

Upon stopping metabolic processes, some tardigrades can undergo anhydrobiosis. Secretory abundant heat-soluble (SAHS) proteins have been reported as candidates for anhydrobiosis-related proteins in tardigrades, which seem to protect extracellular components and/or secretory organelles. We determined structures of a SAHS protein from Ramazzottius varieornatus (RvSAHS1), which is one of the toughest tardigrades. RvSAHS1 shows a β-barrel structure similar to fatty acid-binding proteins (FABPs), in which hydrophilic residues form peculiar hydrogen bond networks, which would provide RvSAHS1 with better tolerance against dehydration. We identified two putative ligand-binding sites: one that superimposes on those of some FABPs and the other, unique to and conserved in SAHS proteins. These results indicate that SAHS proteins constitute a new FABP family.

Crystal structure of secretory abundant heat soluble protein 4 from one of the toughest “water bears” micro‐animals Ramazzottius Varieornatus

“…B ‐factor values showed that the lid region of Rv SAHS4 was highly flexible (Fig. S1, Supporting Information), as is observed in Rv SAHS1 . The variety of residues and the flexibility in the lid structure may be related to ligand binding.…”

“…The overall structure of Rv SAHS4 shared a typical FABP fold having an antiparallel β‐barrel composed of 10 β‐strands, and a helix‐turn‐helix lid between βA and βB. Because main chain atoms could not form hydrogen bonds between βD and βE, there was a gap as is found in Rv SAHS1 and FABPs . The N‐terminal region of the adjacent molecule was inserted into this gap in the crystal structure [Fig.…”

“…While LBS1 in Rv SAHS1 possesses an endogenous FA molecule originating from Escherichia coli in the crystal structure, no electron density corresponding to an FA molecule was observed around LBS1 in Rv SAHS4. Because Rv SAHS4 was expressed and purified by the same methods as those for Rv SAHS1 and crystallized under a condition (Materials and Methods section) similar to that for Rv SAHS1 [100 m M HEPES pH 7.8, 150 m M MgCl 2 , 1 m M ZnSO 4 , 21% v/v polyethylene glycol (PEG) 600, and 5% v/v 1‐butanol or 3% v/v 2‐propanol at 20°C], the absence of the FA molecule indicates that LBS1 of Rv SAHS4 has lower affinity to FAs than that of Rv SAHS1. To use LBS1 in Rv SAHS4, a dramatic structural change in the helix‐turn‐helix lid may be needed, by which Tyr65 is moved away from LBS1.…”

“…(C), S2, Supporting Information]. In contrast, there are only one or two water molecules around the β‐barrel bottom of Rv SAHS1 . This is because hydrophobic residues in Rv SAHS4 (Leu73, Val86, and Phe155) were substituted by residues forming hydrogen bonds with each other in Rv SAHS1 (His72, His83, and Tyr152).…”

“…One of SAHS proteins from R. varieornatus ( Rv SAHS1) is secreted into the culture medium when it is expressed in human cells; therefore, SAHS proteins are thought to protect extracellular components and/or secretory organelles on anhydrobiosis . A crystal structure of Rv SAHS1 revealed that it has a β‐barrel structure resembling fatty acid binding proteins (FABPs) . Residues found in the β‐barrel of Rv SAHS1 were bulky and hydrophilic, while smaller and/or hydrophobic residues are assembled in FABPs.…”