Streptococcal Scl1 and Scl2 Proteins Form Collagen-like Triple Helices

Abstract

The collagens are a family of animal proteins containing segments of repeated Gly-Xaa-Yaa (GXY) motifs that form a characteristic triple-helical structure. Genes encoding proteins with repeated GXY motifs have also been reported in bacteria and phages; however, it is unclear whether these prokaryotic proteins can form a collagen-like triple-helical structure. Here we used two recently identified streptococcal proteins, Scl1 and Scl2, containing extended GXY sequence repeats as model proteins. First we observed that prior to heat denaturation recombinant Scl proteins migrated as homotrimers in gel electrophoresis with and without SDS. We next showed that the collagen-like domain of Scl is resistant to proteolysis by trypsin. We further showed that circular dichroism spectra of the Scl proteins contained features characteristic of collagen triple helices, including a positive maximum of ellipticity at 220 nm. FurthermorethetriplehelicesofScl1andScl2showedatemperature-dependent unfolding with melting temperatures of 36.4 and 37.6°C, respectively, which resembles those seen for collagens. We finally demonstrated by electron microscopy that the Scl proteins are organized into "lollipoplike" structures, similar to those seen in human proteins with collagenous domains. This implies that the repeated GXY tripeptide motif is a structural indicator of collagenlike triple helices in proteins from such phylogenetically distant sources as bacteria and humans.The collagens comprise a large family of structurally related proteins that are essential building elements of connective tissues in animals (1). In addition to contributing to the structural integrity of the tissue, collagens may also affect cell behavior, e.g. by interacting with specific cellular receptors. All collagens contain a triple-helical domain composed of three left-handed polyproline II-type chains wound around the central axis to form a right-handed superhelix (2). The segments of the collagen polypeptide forming a triple helix contain the repeating amino acid sequence Gly-Xaa-Yaa (GXY) in which a glycine residue occupies every third position, since only glycine is small enough to be accommodated at the center of the triple helix. The three chains are linked by hydrogen bonds between the backbone group, -NH (Gly) and the backbone carbonyl group (X position) of another chain, and by water-mediated interactions. The X and Y positions are frequently occupied by the imino acids proline and hydroxyproline, respectively. Hydroxyprolines are formed by post-translational hydroxylation of proline residues and are important in stabilizing the collagen triple helix. In humans, mutations that affect collagen triple helix formation and fiber assembly have serious pathological consequences often leading to death (3).The vertebrate collagens are classified into 19 types that are grouped in two major categories known as fibrillar (types I-III, V, and XI) and nonfibrillar (IV, VI-X, and XII-XIX) collagens. In addition, a variety of other mammalian proteins have been identif...

The Crystal Structure of the Streptococcal Collagen-like Protein 2 Globular Domain from Invasive M3-type Group A Streptococcus Shows Significant Similarity to Immunomodulatory HIV Protein gp41

“…However, the observation that the recombinant CL domain of Scl1 is expressed as a stable triple helix (16,23) contrasts with this hypothesis at least in vivo. Scls have characteristic "lollipop-shaped" domain organization, which seems apt for ligand binding.…”

“…Scls have characteristic "lollipop-shaped" domain organization, which seems apt for ligand binding. Indeed, antibody mapping and electron microscopy imaging analyses confirmed that the stalk-forming CL region projects the globular V region away from the bacterial surface (16), a feature that may facilitate interactions of V regions with their potential targets. Several biologically relevant V region ligands have been identified using experimental approaches.…”

“…Protein Methods-Recombinant Scl2.3-V region polypeptide, designated rScl2.3-V, was generated using the Strep-tag II expression and purification system (IBA GmbH) as described previously (16). Briefly, the 5Ј-portion of the scl2.3 allele from strain MGAS315 encoding the V region of the presumed mature Scl2.3 protein was PCR-amplified using scl2-M3VF (5Ј-GAGATGGCCGATGGTGAAGATGCCCAAAAAAG) forward primer and scl2-M3VR (5Ј-CAGCGTCTCAGCGCTATCAAG-GACATGATCTTGTATGCC) reverse primer and subsequently cloned into the E. coli expression vector pASK-IBA2, generating plasmid pSL155.…”

Definition of the Native and Denatured Type II Collagen Binding Site for Fibronectin Using a Recombinant Collagen System

“…collagen-like protein in S. pyogenes (Scl2.28) contains an N-terminal trimerization domain (V domain) followed by a CL domain with 79 Gly-Xaa-Yaa triplets (22,26,27). A construct in which the collagen domain was duplicated, VCLCL, was cloned and has been used in this study as the host for human collagen sequence insertions that may mediate binding to Fn.…”

“…Here, a recombinant bacterial collagen-like protein system is employed to characterize the amino acid sequences in native and denatured collagen required for Fn binding. A collagen-like protein found in Streptococcus pyogenes, which is readily expressed and easily modified in Escherichia coli, was previously shown to have a triple helix structure and stability similar to that of human fibrillar collagens, even though post-translationally formed hydroxyproline (Hyp) is absent (22)(23)(24). Chimeric collagen-like proteins were generated by inserting potential Fn binding sequences from human collagen type II between bacterial collagen modules.…”