Combining in situ proteolysis and mass spectrometry to crystallize Escherichia coli PgaB

Program in Molecular Structure and Function, The Hospital for Sick Children, 555 University Avenue, Toronto, ON M5G 1X8, Canada.
Acta Crystallographica Section F Structural Biology and Crystallization Communications (Impact Factor: 0.53). 07/2012; 68(Pt 7):842-5. DOI: 10.1107/S1744309112022075
Source: PubMed


The periplasmic poly-β-1,6-N-acetyl-D-glucosamine (PNAG) de-N-acetylase PgaB from Escherichia coli was overexpressed and purified, but was recalcitrant to crystallization. Use of the in situ proteolysis technique produced crystals of PgaB, but these crystals could not be optimized for diffraction studies. By analyzing the initial crystal hits using SDS-PAGE and mass spectrometry, the boundaries of the protein species that crystallized were determined. The re-engineered protein target crystallized reproducibly without the addition of protease and with significantly increased crystal quality. Crystals of the selenomethionine-incorporated protein exhibited the symmetry of space group P2(1)2(1)2(1) and diffracted to 2.1 Å resolution.

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    ABSTRACT: Exopolysaccharides are required for the development and integrity of biofilms produced by a wide variety of bacteria. In Escherichia coli, partial de-N-acetylation of the exopolysaccharide poly-β-1,6-N-acetyl-d-glucosamine (PNAG) by the periplasmic protein PgaB is required for polysaccharide intercellular adhesin-dependent biofilm formation. To understand the molecular basis for PNAG de-N-acetylation, the structure of PgaB in complex with Ni(2+) and Fe(3+) have been determined to 1.9 and 2.1 Å resolution, respectively, and its activity on β-1,6-GlcNAc oligomers has been characterized. The structure of PgaB reveals two (β/α)(x) barrel domains: a metal-binding de-N-acetylase that is a member of the family 4 carbohydrate esterases (CE4s) and a domain structurally similar to glycoside hydrolases. PgaB displays de-N-acetylase activity on β-1,6-GlcNAc oligomers but not on the β-1,4-(GlcNAc)(4) oligomer chitotetraose and is the first CE4 member to exhibit this substrate specificity. De-N-acetylation occurs in a length-dependent manor, and specificity is observed for the position of de-N-acetylation. A key aspartic acid involved in de-N-acetylation, normally seen in other CE4s, is missing in PgaB, suggesting that the activity of PgaB is attenuated to maintain the low levels of de-N-acetylation of PNAG observed in vivo. The metal dependence of PgaB is different from most CE4s, because PgaB shows increased rates of de-N-acetylation with Co(2+) and Ni(2+) under aerobic conditions, and Co(2+), Ni(2+) and Fe(2+) under anaerobic conditions, but decreased activity with Zn(2+). The work presented herein will guide inhibitor design to combat biofilm formation by E. coli and potentially a wide range of medically relevant bacteria producing polysaccharide intercellular adhesin-dependent biofilms.
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    No preview · Article · Mar 2014 · Methods in molecular biology (Clifton, N.J.)
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