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. 2018 Oct 9;57(40):5864-5876.
doi: 10.1021/acs.biochem.8b00783. Epub 2018 Sep 25.

Differential Substrate Recognition by Maltose Binding Proteins Influenced by Structure and Dynamics

Shantanu Shukla  1   2 , Khushboo Bafna  1 , Caeley Gullett  2 , Dean A A Myles  1   2 , Pratul K Agarwal  3 , Matthew J Cuneo  2   4
Affiliations

Differential Substrate Recognition by Maltose Binding Proteins Influenced by Structure and Dynamics

Shantanu Shukla et al. Biochemistry. .

Abstract

The genome of the hyperthermophile Thermotoga maritima contains three isoforms of maltose binding protein (MBP) that are high-affinity receptors for di-, tri-, and tetrasaccharides. Two of these proteins (tmMBP1 and tmMBP2) share significant sequence identity, approximately 90%, while the third (tmMBP3) shares less than 40% identity. MBP from Escherichia coli (ecMBP) shares 35% sequence identity with the tmMBPs. This subset of MBP isoforms offers an interesting opportunity to investigate the mechanisms underlying the evolution of substrate specificity and affinity profiles in a genome where redundant MBP genes are present. In this study, the X-ray crystal structures of tmMBP1, tmMBP2, and tmMBP3 are reported in the absence and presence of oligosaccharides. tmMBP1 and tmMBP2 have binding pockets that are larger than that of tmMBP3, enabling them to bind to larger substrates, while tmMBP1 and tmMBP2 also undergo substrate-induced hinge bending motions (∼52°) that are larger than that of tmMBP3 (∼35°). Small-angle X-ray scattering was used to compare protein behavior in solution, and computer simulations provided insights into dynamics of these proteins. Comparing quantitative protein-substrate interactions and dynamical properties of tmMBPs with those of the promiscuous ecMBP and disaccharide selective Thermococcus litoralis MBP provides insights into the features that enable selective binding. Collectively, the results provide insights into how the structure and dynamics of tmMBP homologues enable them to differentiate between a myriad of chemical entities while maintaining their common fold.

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Conflict of interest statement

The authors declare no competing financial interest.

Figures

Figure 1:
Figure 1:. Structural details of the three tmMBP isoforms in complex with maltotetraose and maltose.
(A) Comparison of apo (gray) and maltotetraose bound (red) structures of tmMBP1(left) and the structural details of the substrate interaction in the binding pocket (right). (B) Apo and maltotetraose bound structures of tmMBP2. (C) Apo and maltose bound structures of tmMBP3. In the left panels, substrate is shown as green sticks. In the right panels, the ligands are shown as blue sticks and protein residues as green (hydrophobic contact with substrates) and gray (hydrophilic contact with substrates) stick representation. Direct hydrogen bonds between the protein and the ligands are shown as black dashed lines; magenta spheres indicate oxygen from water molecules observed in crystal structures; and water mediated H-bonds to the ligands as red dashed lines and indirect water mediated H-bonds with binding site residues (cyan sticks) as yellow dashed lines. Some residues making two different types of interactions include: hydrophobic and indirect H-bonds (F41 of tmMBP1); hydrophobic and direct H-bonds (W67 of tmMBP1 and W296 of tmMBP3); and direct and indirect H-bonds (E32 and D133 of tmMBP3).
Figure 2:
Figure 2:. Differential binding of tetra- and di-saccharides in MBPs.
(A) Maltotetraose bound to tmMP1, tmMBP2, and ecMBP. (B) Maltose bound to tmMBP3, trehalose bound to tlMBP and maltose bound to ecMBP. The bound substrates are shown in blue sticks and protein residues are shown as green (hydrophobic contact with substrates) and gray (hydrophilic contact with substrates) sticks. Sub-sites in the binding pocket (S1, S2, S3 and S4) are separated by gray vertical dashed lines. Loop 1 (L1), and helices H1, H2 and H3 are also marked for each complex.
Figure 3:
Figure 3:. Radius of gyration (Rg) computed from MD simulations.
Panels A-D show tmMBP1 results for MAL1, MAL2, MTR and MTR; panels E-H show tmMBP2 results for MAL1, MAL2, MTR and MTR; and panels I-K show tmMB1 results for GLU1, GLU2, and MAL. Rg for apo simulations depicted in red and the substrate bound simulations in blue, and average value of Rg computed from all simulation snapshots are shown by horizontal lines. MAL1 and MAL2 indicate simulations with maltose starting in two alternate positions; site S1+S2 for MAL1 and S2+S3 for MAL2. GLU1 simulation started with glucose in S1 binding pocket and GLU2 in the S2 binding pocket.
Figure 4:
Figure 4:. Comparison of tmMBP1 and tmMBP3 conformational flexibility based on RMSF10.
(A) tmMBP1 and (B) tmMBP3. The tube thickness corresponds to degree of flexibility, with thicker tubes (green/yellow) indicating more flexible regions than the rest of rigid protein (dark blue). Surface exposed regions displaying higher than average conformational flexibility are marked by black ellipses (40–60, 130–150, and 165–195 for tmMBP1 and 50–85, 155–175, and 197–211 for tmMBP3), and the plots below compare the observed values for apo and protein in complex with various substrates. The dashed red line in the plots indicate the average value of RMSF10 observed in the apo proteins for comparison.
Figure 5:
Figure 5:. Protein-substrate interaction energy.
The interaction energy was computed as a sum of electrostatics and van der Waals contributions between each protein residue and substrate unit pair. The yellow-green-blue areas with more favorable contacts are highlighted, and the corresponding protein residue is marked. See Figure 3 legend in main manuscript for the substrate key.
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