Crystallization and preliminary X-ray analysis of mannosyl-3-phosphoglycerate phosphatase from Thermus thermophilus HB27

Abstract

Mannosylglycerate (MG) is primarily known as an osmolyte and is widely distributed among (hyper)thermophilic marine microorganisms. The synthesis of MG via mannosyl-3-phosphoglycerate synthase (MpgS) and mannosyl-3-phosphoglycerate phosphatase (MpgP), the so-called two-step pathway, is the most prevalent route among these organisms. The phosphorylated intermediate mannosyl-3-phosphoglycerate is synthesized by the first enzyme and is subsequently dephosphorylated by the second. The structure of MpgS from the thermophilic bacterium Thermus thermophilus HB27 has recently been solved and characterized. Here, the cloning, expression, purification, crystallization and preliminary crystallographic analysis of MpgP from T. thermophilus HB27 are reported. Size-exclusion chromatography assays suggested a dimeric assembly in solution for MpgP at pH 6.3 and together with differential scanning fluorimetry data showed that high ionic strength and charge compensation were required to produce a highly pure and soluble protein sample for crystallographic studies. The crystals obtained belonged to the monoclinic space group P2(1), with unit-cell parameters a=39.52, b=70.68, c=95.42 Å, β=92.95°. Diffraction data were measured to 1.9 Å resolution. Matthews coefficient calculations suggested the presence of two MpgP monomers in the asymmetric unit and the calculation of a self-rotation Patterson map indicated that the two monomers could be related by a noncrystallographic twofold rotation axis, forming a dimer.

Figure 1

The two pathways for the synthesis of mannosylglycerate. Legend: GDP-Man, GDP-α-d-mannose; 3-PG, 3-d-phosphoglycerate; D-Gly, d-glycerate; MG, α-d-mannosylglycerate; P_i, inorganic phosphate; MpgS, mannosyl-3-phosphoglycerate synthase (EC 2.4.1.217); MpgP, mannosyl-3-phosphoglycerate phosphatase (EC 3.1.3.70); MgS, mannosylglycerate synthase (EC 2.4.1.–). Originally published in Gonçalves et al. (2010 ▶). Reproduced with permission from the American Society for Biochemistry and Molecular Biology.

Figure 2

(a) 15% SDS–PAGE of pure recombinant T. thermophilus HB27 MpgP. Lane 1, Low Range Molecular Mass Markers (Bio-Rad) consisting of the protein markers phosphorylase b (97.4 kDa), bovine serum albumin (66.2 kDa), ovalbumin (43 kDa), carbonic anhydrase (31 kDa), soybean trypsin inhibitor (21.5 kDa) and lysozyme (14.4 kDa). Lane 2, MpgP monomer migrating according to its molecular mass (28.18 kDa). (b) Elution profile of MpgP loaded onto an analytical 2.4 ml Superdex 200 3.2/30 precision column. The MpgP elution profile is represented as a full curve in blue. Elution profiles of protein standards are shown as dashed curves for comparison. In addition to their standard elution peaks, degradation products are also eluted. mAU, milliunits of absorption.

Figure 3

DSF thermal denaturation curves for MpgP. The buffer composition does not take into account the 10% dilution effect during protein-sample dilution (see §2). (a) MpgP stability as a function of pH at high ionic strength. The thermal unfolding profile is shown only within the pH interval 5.0–7.0 as the protein was less stable at other pH values. The chemical nature of the buffer also had an effect on the stability of MpgP as observed for the profiles between curves F01 and F03. (b) MpgP stability as function of salt. In the MES buffer system the stability of MpgP increases significantly with an increase in ionic strength (curves A10, C10 and E10), while the substitution of equivalent amounts of NaCl by KCl have little effect (curves C10 and G10). Sodim citrate pH 5.5 buffer (curves A07, C07, E07 and G07) was an exception since MpgP exhibited similar unfolding profiles regardless of the presence of salt.

Figure 4

MpgP elution profiles from the SEC analysis with a Superdex 75 3.2/30 precision column. MpgP elution profiles are represented as full curves. The upper left text in each panel indicates the gel-filtration buffer composition in which MpgP was eluted and is colour-coded according to the colours of the respective MpgP elution curves. MES–NaOH pH 6.3 buffer was used in all gel-filtration runs. (a) Effect of NaCl in the elution of MpgP. The elution profiles of protein standards are shown as dashed curves for comparison. In addition to their standard elution peaks, lower molecular-mass degradation products are also eluted. (b) Effect of EDTA and DTT on the MpgP elution profile. mAU, milliunits of absorption.

Figure 5

Native MpgP crystals used for data collection.

Figure 6

Self-rotation Patterson map in the κ = 180° section, calculated with MOLREP from CCP4 (Collaborative Computational Project, Number 4, 1994 ▶) using default parameters, with a radius of integration of 19.8 Å and data in the resolution range 39.5 > d > 3.0 Å. The unique b axis in space group P2₁ is oriented along the y axis in the figure. Two equivalent noncrystallographic symmetry peaks (NCS) are observed perpendicular to the crystallographic b axis and with an intensity of 68% relative to the origin, suggesting the presence of an MpgP dimer in the asymmetric unit, with the two monomers related by twofold NCS.