Journal of Molecular Biology
Structure and Function Characterization of the a1a2 Motifs of Streptococcus pyogenes M Protein in Human Plasminogen Binding
Graphical Abstract
Introduction
Group A streptococcus (GAS) is a human pathogen that causes a range of clinical conditions from superficial skin and pharyngeal infections to much more serious conditions such as rheumatic fever, necrotizing fasciitis, and streptococcal toxic shock [1]. There are over 600 million cases of GAS infection reported each year, which pose significant global economic and healthcare burdens [2]. GAS expresses a number of virulence factors to colonize host tissues [3], disseminate from primary infection sites [4], [5], and evade host immune surveillance [3], [6]. One of the key virulence determinants is the surface-bound M protein [7], [8].
M proteins are typically α-helical coiled-coil proteins consisting of a hypervariable N-terminal region, followed by variable A and B repeat domains, and the conserved C repeats and D domain [9]. There are more than 250 M proteins documented to date via sequence analysis of the hyper-variable N-terminal region [10]. Plasminogen-binding group A streptococcal M-like protein (PAM) belongs to a subgroup of the M protein family with the Plg binding a1a2 repeat located at the variable A domain [11]. PAM on the bacterial surface recruits plasminogen (Plg) and facilitates the formation of plasmin (Plm) by streptokinase, a plasminogen activator also secreted by GAS, thus enabling the pathogen to disseminate from the primary infection site and recolonize within the host [12].
Plg, the inactive precursor of the protease Plm [13], [14], is a 92-kDa multi-domain glycoprotein. It consists of seven domains, an N-terminal PAN-apple (PAp) domain followed by five homologous kringle (KR1–5) domains, and a C-terminal serine protease (SP) domain. All KR domains, except for KR3, have a functional lysine-binding site (LBS: Asp-X-Asp for KR1, 4–5, and Asp-Arg-Glu for KR2) that binds to C-terminal lysine or internal lysine or arginine residues present on target substrates or receptors [14]. Previous crystallographic and NMR studies on VEK30, an internal peptide with the a1 repeat from PAM53 (GAS strain AP53, Fig. 1), revealed that it binds to the lysine binding site (LBS) of Plg KR2 via an internal lysine isostere site on the peptide [15], [16], [17]. Specifically, VEK30 binds to KR2 via the key residues Arg101VEK30 and His102VEK30 (hence, it is also called the RH motif, Fig. 1) [15], [16], [18]. Intriguingly, PAM53, as well as many PAM variants [19], has a second RH motif (RH2) located at the a2 repeat. A recent study on a peptide called VKK38 (isolated from GAS strain NS455, Fig. 1), which contains both the a1 and a2 repeats with a 3-residue insertion (Val109–His110–Asp111) between the repeats, suggested that the RH2 motif is also a KR2 binder [20] and that both RH motifs are located at the disordered regions of the apo-peptide. Using combined analytical ultracentrifugation, isothermal titration calorimetry, and NMR, this study further suggested that VKK38 binds to two KR2 domains in solution. Exactly how the RH motifs interact with two KR2 domains simultaneously and their molecular interaction remain to be fully elucidated experimentally.
In the present study, we report a 1.7-Å co-crystal structure of VEK75 (an a1a2 containing peptide from PAM53 residues 76–150 [21]; Fig. 1) simultaneously bound to two molecules of KR2, which contains its native LBS motif, confirming the aforementioned VKK38 data. We further characterize the functional and binding affinity of RH1 and RH2 for Plg using enzyme kinetics and surface plasmon resonance (SPR) studies on the RH deletion mutants. Our results reveal unexpectedly that RH2 has a higher affinity for Plg than RH1, suggesting that a1 and a2 might play a different role in the final PAM-Plg binary complex.
Section snippets
Stoichiometry of VEK75–KR2 complex
Size exclusion chromatography (AdvanceBio SEC 300 Å column; Agilent) combined with multi-angle light scattering (SEC-MALS) was used to determine the molecular masses of VEK75, KR2, and VEK75–KR2 complex in solution. The SEC-MALS studies reveal that VEK75 is a monomer in solution (8.94 ± 0.89 kDa; Table 1 and Fig. 2) instead of a weakly associated dimer as reported previously [21]; KR2 is also a monomer in solution (13.09 ± 0.79 kDa) as expected. The molar mass of VEK75–KR2 complex is
Discussion
PAM is a key virulence factor of skin-tropic GAS strains and also the highest-affinity Plg receptor described [20], [22]. Despite its pathological significance, the structural details of the full-length PAM and how it binds to Plg are not known. Prior to this study, the binding of a1 to KR2 has been well documented [15], [16], [23], [24]; however, the molecular interactions between a2 and KR2 have not been defined in detail experimentally.
The x-ray crystal structure provides the atomic details
Mutagenesis and protein purification
Recombinant VEK75 fused to the B1 domain of streptococcal G protein (GB1) is expressed using the pET-15b plasmid (Fig. S6a). RH1 and RH2 mutations were introduced using primers listed in Table S3 and a standard Quikchange site-directed mutagenesis protocol. Using RH1 mutant (VEK75ΔRH1) as the DNA template, RH2 mutation was introduced to obtain VEK75ΔRH1/RH2. Mutations were verified by Sanger sequencing (Micromon DNA Sequencing Facility, Monash University). VEK75 and mutants were expressed in
Acknowledgments
The authors thank the Australian Synchrotron for MX2 beamtime and technical assistance, and the Monash Molecular Crystallization Facility for setting up crystallization experiments. This research was undertaken in part using the MX2 beamline at the Australian Synchrotron, part of ANSTO, and made use of the ACRF detector. This work was supported in part by the Australian National Health Medical Research Council. G.W. and B.A.M. are supported by Monash University PhD scholarships, and J.C.W. is
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Contributed equally.