Structural and Functional Insights into Aeropyrum pernix OppA, a Member of a Novel Archaeal OppA Subfamily

Abstract

In this study we gain insight into the structural and functional characterization of the Aeropyrum pernix oligopeptide-binding protein (OppA Ap ) previously identified from the extracellular medium of an Aeropyrum pernix cell culture at late stationary phase. OppA Ap showed an N-terminal Q32 in a pyroglutamate form and C-terminal processing at the level of a threonine-rich region probably involved in protein membrane anchoring. Moreover, the OppA Ap protein released into the medium was identified as a "nicked" form composed of two tightly associated fragments detachable only under strong denaturing conditions. The cleavage site E569-G570 seems be located on an exposed surface loop that is highly conserved in several three-dimensional (3D) structures of dipeptide/oligopeptide-binding proteins from different sources. Structural and biochemical properties of the nicked protein were virtually indistinguishable from those of the intact form. Indeed, studies of the entire bacterially expressed OppA Ap protein owning the same N and C termini of the nicked form supported these findings. Moreover, in the middle exponential growth phase, OppA Ap was found as an intact cell membrane-associated protein. Interestingly, the native exoprotein OppA Ap was copurified with a hexapeptide (EKFKIV) showing both lysines methylated and possibly originating from an A. pernix endogenous stress-induced lipoprotein. Therefore, the involvement of OppA Ap in the recycling of endogenous proteins was suggested to be a potential physiological function. Finally, a new OppA from Sulfolobus solfataricus, SSO1288, was purified and preliminarily characterized, allowing the identification of a common structural/genetic organization shared by all "true" archaeal OppA proteins of the dipeptide/oligopeptide class.

A shift between mineral and nonmineral sources of iron and sulfur causes proteome-wide changes in Methanosarcina barkeri

“…The difference in sequence could mean that the proteins have different oligopeptide ligands. Oppa is usually lipid anchored in the membrane, but studies have reported that a soluble form actively binds peptides in the cytosol of other organisms ( 26 ). Our collaborators have previously noted that Methanococcus voltae (a close cousin to M. barkeri ) cells grown on FeS 2 show increased biofilm formation and clumping ( 14 ), and the proteomes of Fe(II)/HS − - and FeS 2 -grown M. barkeri have upregulated proteins related to biofilm formation and signaling.…”

Structural basis for peptide recognition by archaeal oligopeptide permease A

“…Then, the solution was mixed with the peptide bradykinin (RPPGFSPFR) and crystallized in order to elucidate interactions with the specific peptide rather than endogenous peptides. Bradykinin was used in peptide selectivity studies of other archaeal OppAs 16,17 …”

“…Bradykinin was used in peptide selectivity studies of other archaeal OppAs. 16,17 The structure determination by the molecular replacement method using the structure of B. subtilis OppA (PDB ID: 1XOC) 25 was unsuccessful due to low sequence identity of 14%. Structure determination by multi-wavelength anomalous dispersion or singlewavelength anomalous dispersion (SAD) methods using selenomethionine (SeMet) derivative protein was unsuccessful because most of the methionine residues may be located in the loop region according to secondary-structure prediction.…”

“…Based on a genomic survey of Pyrococcus species, archaeal Opps consist of five components similarly to bacterial ones 3 . Archaeal OppA was first isolated and characterized from the extracellular medium of Aeropyrum pernix 15 and functionally characterized by assays of peptide binding and selectivity using OppAs of A. pernix 16 and Sulfolobus solfataricus 17 . There are several reports that archaea produce membrane vesicles.…”

Diversity and Subcellular Distribution of Archaeal Secreted Proteins

“…The class of oligopeptide-binding proteins characterized in the crenarchaeal species S. solfataricus and Aeropyrum pernix contains an N-terminal secretory signal sequence with an SPase I cleavage site and an additional C-terminal transmembrane segment (Elferink et al, 2001 ; Gogliettino et al, 2010 ; Balestrieri et al, 2011 ). The membrane anchor is preceded by a stretch of glycosylated serine and threonine residues.…”