Structural-Functional Analysis Reveals a Specific Domain Organization in Family GH20 Hexosaminidases

Abstract

Hexosaminidases are involved in important biological processes catalyzing the hydrolysis of N-acetyl-hexosaminyl residues in glycosaminoglycans and glycoconjugates. The GH20 enzymes present diverse domain organizations for which we propose two minimal model architectures: Model A containing at least a non-catalytic GH20b domain and the catalytic one (GH20) always accompanied with an extra α-helix (GH20b-GH20-α), and Model B with only the catalytic GH20 domain. The large Bifidobacterium bifidum lacto-N-biosidase was used as a model protein to evaluate the minimal functional unit due to its interest and structural complexity. By expressing different truncated forms of this enzyme, we show that Model A architectures cannot be reduced to Model B. In particular, there are two structural requirements general to GH20 enzymes with Model A architecture. First, the non-catalytic domain GH20b at the N-terminus of the catalytic GH20 domain is required for expression and seems to stabilize it. Second, the substrate-binding cavity at the GH20 domain always involves a remote element provided by a long loop from the catalytic domain itself or, when this loop is short, by an element from another domain of the multidomain structure or from the dimeric partner. Particularly, the lacto-N-biosidase requires GH20b and the lectin-like domain at the N- and C-termini of the catalytic GH20 domain to be fully soluble and functional. The lectin domain provides this remote element to the active site. We demonstrate restoration of activity of the inactive GH20b-GH20-α construct (model A architecture) by a complementation assay with the lectin-like domain. The engineering of minimal functional units of multidomain GH20 enzymes must consider these structural requirements.

Fig 1. Hydrolytic reaction of lacto-N-tetraose catalyzed by B. bifidum lacto-N-biosidase.

Fig 2. Crystal structures of GH20 β-N-acetylhexosaminidases.

(A) Structures according to model A architecture. (B) Structures according to model B architecture. GH20b domains are colored in orange, GH20 domains in blue, α-helix in green, and the rest of accompanying domains in yellow and purple. Loop 2 from GH20 domain is in red. Dimer counterparts follow the same coloring scheme but with lower intensity.

Fig 3. Truncated forms of B.bifidum Lacto-N-biosidase.

Fig 4. Size exclusion chromatography of the full length protein and different constructs of Lacto-N-biosidase from B.bifidum.

(A) Chromatogram of the full length protein (FL) and constructs A, D and F. (B) SDS-PAGE of the eluted proteins. M: broad range protein molecular weight marker (200, 116.2, 97.4, 66.2, 45, 31 kDa).

Fig 5. Remote elements and their key residues that complement the active site of GH20 β-N-acetylhexosaminidases.

(A) B. bifidum lacto-N-biosidase. (B) S. plicatus. (C) S. gordonii N-acetylhexosaminidases. Dot lines represent interactions between residues of the remote element and the active site as indicated in Table 2.

Fig 6. Multiple sequence alignment of β-N-acetylhexosaminidases of known structure from family GH20.

Domains distribution is shown as colored boxes on top. Accompanying domains are not shown. Conserved arginine and glutamate/aspartate sites are marked with a star. Sequences were downloaded from UniProt entry names: SpHex (O85361_STRPL), ScHex (Q9L068), Bf3009 (Q5LAT3) NahA (A1RBZ5_ARTAT), Hex1T (D2KW09_9BACL), SmCHB (CHB_SERMA), LnbB (B3TLD6_BIFBI), HexA (HEXA_HUMAN), HexB (HEXB_HUMAN), OfHex1 (Q06GJ0_OSTFU), GcnA (Q6ST21_STRGN), DspB (Q840G9_AGGAC), StrH_R6 (Q8DRL6_STRR6), StrH_TIGR4 (STRH_STRPN). Alignment was performed with PROMALS [17]. Conserved positions are coloured according to ClustalW colour scheme.

Fig 7. Complementation assay of construct A with construct F at different molar ratios.

Conditions: 50 nM construct A, 25 nM-500 nM construct F, 0.25 mM p-nitrophenyl β-lacto-N-bioside, 25 mM citrate-25 mM phosphate buffer, pH 4.5, 30°C. The relative standard deviation did not overcome 2% except at 6 and 10 molar ratio that was lower than 15%.

Fig 8. Size exclusion chromatography of the complementation assay in a ratio 1:6 of constructs A and F.

(A) Chromatogram of the eluted fractions: absorbance at 280nm (-), hydrolase activity (•••). (B) SDS-PAGE of different fractions. M: broad range protein molecular weight marker (200, 116.2, 97.4, 66.2, 45, 31 kDa); A: construct A Standard, fractions C1–C6, fractions L1–L2. The relative standard deviations for the molecular weights and activities were less than 2% and 15% respectively.