Abstract
For a subset of pathogenic microorganisms, including Streptococcus pneumoniae, the recognition and degradation of host hyaluronan contributes to bacterial spreading through the extracellular matrix and enhancing access to host cell surfaces. The hyaluronate lyase (Hyl) presented on the surface of S. pneumoniae performs this role. Using glycan microarray screening, affinity electrophoresis, and isothermal titration calorimetry we show that the N-terminal module of Hyl is a hyaluronan-specific carbohydrate-binding module (CBM) and the founding member of CBM family 70. The 1.2 Å resolution x-ray crystal structure of CBM70 revealed it to have a β-sandwich fold, similar to other CBMs. The electrostatic properties of the binding site, which was identified by site-directed mutagenesis, are distinct from other CBMs and complementary to its acidic ligand, hyaluronan. Dynamic light scattering and solution small angle x-ray scattering revealed the full-length Hyl protein to exist as a monomer/dimer mixture in solution. Through a detailed analysis of the small angle x-ray scattering data, we report the pseudoatomic solution structures of the monomer and dimer forms of the full-length multimodular Hyl.
Highlights
hyaluronate lyase (Hyl) from Streptococcus pneumoniae degrades host hyaluronan, an important polysaccharide component of the mammalian extracellular matrix
The surface of S. pneumoniae is a complex landscape of macromolecules that, in many cases, help to mediate the interaction of the bacterium with the host
In SpuA, one of the CBM41 modules closes over the active site, aiding directly in substrate recognition, whereas a second module is oriented away from the active site and cell surface to presumably function in adherence to glycogen [52]
Summary
Hyl from Streptococcus pneumoniae degrades host hyaluronan, an important polysaccharide component of the mammalian extracellular matrix. SpCBM70 SpCBM70 SpCBM70C, CBM mutants SpCBM70C, CBM mutants Hyl W82A W82A R112A R112A H116A H116A K143A K143A R145A R145A K185A K185A PL8 PL8 tribution of Hyl to virulence is not significant; the production of Hyl by the bacterium has a strong potentiating effect on the critical virulence factor pneumolysin, a poreforming cytotoxin that binds to host cholesterol [8, 9] This potentiating effect is consistent with Hyl playing a role in breaking the integrity of the extracellular matrix barrier, thereby providing improved access of the bacterium and pneumolysin to the host cell surface. There is comparatively little information regarding the molecular recognition of hyaluronan by pathogenic microbes To address this knowledge gap and test the hypothesis that the N-terminal module of Hyl is a hyaluronan-specific CBM, we examined the structural and functional properties of this module using a combination of approaches, including glycan microarray, isothermal titration calorimetry, x-ray crystallography, and sitedirected mutagenesis. The SAXS-based model of Hyl, interpreted on the basis of its surface presentation, suggests a conformation optimized for the interaction of the CBM with hyaluronan present in the extracellular matrix
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