Unravelling the Carbohydrate-Binding Preferences of the Carbohydrate-Binding Modules of AMP-Activated Protein Kinase

作者：Mobbs Jesse I; Di Paolo Alex; Metcalfe Riley D; Selig Emily; Stapleton David I; Griffin Michael D W; Gooley Paul R^*

来源：ChemBioChem, 2018, 19(3): 229-238.

DOI：10.1002/cbic.201700589

• carbohydrate binding
• mutagenesis
• proteins
• structure elucidation
• thermodynamics

The beta subunit of adenosine monophosphate (AMP)-activated protein kinase (AMPK), which exists as two isoforms (beta 1 and beta 2) in humans, has a carbohydrate-binding module (CBM) that interacts with glycogen. Although the beta 1- and beta 2-CBMs are structurally similar, with strictly conserved ligand-contact residues, they show different carbohydrate affinities. beta 2-CBM shows the strongest affinity for both branched and unbranched oligosaccharides and it has recently been shown that a Thr insertion into beta 2-CBM (Thr101) forms a pocket to accommodate branches. This insertion does not explain why beta 2-CBM binds all carbohydrates with stronger affinity. Herein, it is shown that residue 134 (Val for beta 2 and Thr for beta 1), which does not come into contact with a carbohydrate, appears to account for the affinity difference. Characterisation by NMR spectroscopy, however, suggests that mutant beta 2-Thr101 Delta/Val134Thr differs from that of beta 1-CBM, and mutant beta 1Thr101ins/Thr134Val differs from that of beta 2-CBM. Furthermore, these mutants are less stable to chemical denaturation, relative to that of wild-type beta-CBMs, which confounds the affinity analyses. To support the importance of Thr101 and Val134, the ancestral CBM has been constructed. This CBM retains Thr101 and Val134, which suggests that the extant beta 1-CBM has a modest loss of function in carbohydrate binding. Because the ancestor bound carbohydrate with equal affinity to that of beta 2CBM, it is concluded that residue 134 plays an indirect role in carbohydrate binding.

• 出版日期2018-2-2