Inhibitor binding mode and allosteric regulation of Na+-glucose symporters

Lon belongs to a unique group of AAA+proteases that bind DNA. However, the DNA-mediated regulation of Lon remains elusive. Here, the crystal structure of the α subdomain of the Lon protease fromBrevibacillus thermoruber(Bt-Lon) is presented, together with biochemical data, and the DNA-binding mode is delineated, showing that Arg518, Arg557 and Arg566 play a crucial role in DNA binding. Electrostatic interactions contributed by arginine residues in the AAA+module are suggested to be important to DNA binding and allosteric regulation of enzymatic activities. Intriguingly, Arg557, which directly binds DNA in the α subdomain, has a dual role in the negative regulation of ATPase stimulation by DNA and in the domain–domain communication in allosteric regulation of Bt-Lon by substrate. In conclusion, structural and biochemical evidence is provided to show that electrostatic interaction in the AAA+module is important for DNA binding by Lon and allosteric regulation of its enzymatic activities by DNA and substrate.

Download Full-text

Structural Comparison, Substrate Specificity, and Inhibitor Binding of AGPase Small Subunit from Monocot and Dicot: Present Insight and Future Potential

BioMed Research International ◽

10.1155/2014/583606 ◽

2014 ◽

Vol 2014 ◽

pp. 1-20 ◽

Cited By ~ 5

Author(s):

Kishore Sarma ◽

Priyabrata Sen ◽

Madhumita Barooah ◽

Manabendra D. Choudhury ◽

Shubhadeep Roychoudhury ◽

...

Keyword(s):

Catalytic Mechanism ◽

Function Analysis ◽

Allosteric Regulation ◽

Binding Specificity ◽

Small Subunit ◽

Crop Plants ◽

Site Directed Mutagenesis ◽

Dynamics Simulation ◽

Dimensional Structure ◽

Inhibitor Binding

ADP-glucose pyrophosphorylase (AGPase) is the first rate limiting enzyme of starch biosynthesis pathway and has been exploited as the target for greater starch yield in several plants. The structure-function analysis and substrate binding specificity of AGPase have provided enormous potential for understanding the role of specific amino acid or motifs responsible for allosteric regulation and catalytic mechanisms, which facilitate the engineering of AGPases. We report the three-dimensional structure, substrate, and inhibitor binding specificity of AGPase small subunit from different monocot and dicot crop plants. Both monocot and dicot subunits were found to exploit similar interactions with the substrate and inhibitor molecule as in the case of their closest homologue potato tuber AGPase small subunit. Comparative sequence and structural analysis followed by molecular docking and electrostatic surface potential analysis reveal that rearrangements of secondary structure elements, substrate, and inhibitor binding residues are strongly conserved and follow common folding pattern and orientation within monocot and dicot displaying a similar mode of allosteric regulation and catalytic mechanism. The results from this study along with site-directed mutagenesis complemented by molecular dynamics simulation will shed more light on increasing the starch content of crop plants to ensure the food security worldwide.

Download Full-text

DFG-1 residue controls inhibitor binding mode and affinity providing a basis for rational design of kinase inhibitor selectivity

10.1101/2020.07.01.182642 ◽

2020 ◽

Author(s):

Martin Schröder ◽

Alex N. Bullock ◽

Oleg Federov ◽

Franz Bracher ◽

Apirat Chaikuad ◽

...

Keyword(s):

Rational Design ◽

Kinase Inhibitors ◽

Kinase Inhibitor ◽

Binding Mode ◽

Selectivity Filter ◽

Binding Modes ◽

Selective Inhibitors ◽

Inhibitor Binding ◽

Binding Pockets ◽

Inhibitor Selectivity

ABSTRACTSelectivity remains a challenge for ATP-mimetic kinase inhibitors, an issue that may be overcome by targeting unique residues or binding pockets. However, to date only few strategies have been developed. Here we identify that bulky residues located N-terminal to the DFG motif (DFG-1) represent an opportunity for designing highly selective inhibitors with unexpected binding modes. We demonstrate that several diverse inhibitors exerted selective, non-canonical binding modes that exclusively target large hydrophobic DFG-1 residues present in many kinases including PIM, CK1, DAPK and CLK. Using the CLK family as a model, structural and biochemical data revealed that the DFG-1 valine controlled a non-canonical binding mode in CLK1, providing a rational for selectivity over the closely-related CLK3 which harbors a smaller DFG-1 alanine. Our data suggests that targeting the restricted back pocket in the small fraction of kinases that harbor bulky DFG-1 residues offers a versatile selectivity filter for inhibitor design.

Download Full-text