scholarly journals The flexible N-terminus of BchL autoinhibits activity through interaction with its [4Fe-4S] cluster and relieved upon ATP binding

2020 ◽  
pp. jbc.RA120.016278
Author(s):  
Elliot I Corless ◽  
Syed Muhammad Saad Imran ◽  
Maxwell B Watkins ◽  
John-Paul Bacik ◽  
Jenna Mattice ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Local Environment ◽  
Binding Pocket ◽  
Free Form ◽  
Atp Binding ◽  
Atp Binding Site ◽  
Exchange Mass Spectrometry ◽  
Protochlorophyllide Reduction ◽  
N Terminus ◽  
Poor Tolerance

A key step in bacteriochlorophyll biosynthesis is the reduction of protochlorophyllide to chlorophyllide, catalyzed by dark-operative protochlorophyllide oxidoreductase (DPOR). DPOR contains two [4Fe-4S]-containing component proteins (BchL and BchNB) that assemble upon ATP binding to BchL to coordinate electron transfer and protochlorophyllide reduction. But the precise nature of the ATP-induced conformational changes are poorly understood. We present a crystal structure of BchL in the nucleotide-free form where a conserved, flexible region in the N-terminus masks the [4Fe-4S] cluster at the docking interface between BchL and BchNB. Amino acid substitutions in this region produce a hyper-active enzyme complex, suggesting a role for the N-terminus in auto-inhibition. Hydrogen deuterium exchange mass spectrometry shows that ATP-binding to BchL produces specific conformational changes leading to release of the flexible N-terminus from the docking interface. The release also promotes changes within the local environment surrounding the [4Fe-4S] cluster and promotes BchL complex formation with BchNB. A key patch of amino acids, Asp-Phe-Asp (the ‘DFD patch’), situated at the mouth of the BchL ATP-binding pocket promotes inter-subunit cross stabilization of the two subunits. A linked BchL dimer with one defective ATP-binding site does not support protochlorophyllide reduction, illustrating nucleotide binding to both subunits as a prerequisite for the inter-subunit cross stabilization. The masking of the [4Fe-4S] cluster by the flexible N-terminal region and the associated inhibition of activity is a novel mechanism of regulation in metalloproteins. Such mechanisms are possibly an adaptation to the anaerobic nature of eubacterial cells with poor tolerance for oxygen.

scholarly journals The flexible N-terminus of BchL protects its [4Fe-4S] cluster in oxygenic environments and autoinhibits activity

2019 ◽  
Author(s):  
Elliot Corless ◽  
Syed Muhammad Saad Imran ◽  
Maxwell B. Watkins ◽  
Sofia Origanti ◽  
John-Paul Bacik ◽  
...  
Keyword(s):  
Electron Transfer ◽  
Enzyme Stability ◽  
Binding Pocket ◽  
Free Form ◽  
Release Mechanism ◽  
Atp Binding ◽  
Atp Binding Site ◽  
Substrate Reduction ◽  
Evolutionarily Conserved ◽  
N Terminus

AbstractThe dark-operative protochlorophyllide oxidoreductase (DPOR) enzyme contains two [4Fe-4S]- containing component proteins (BchL and BchNB) that assemble in an ATP-dependent fashion to coordinate electron transfer and reduction of protochlorophyllide to chlorophyllide. Photosynthesis generates an oxygenic environment that is non-optimal for [Fe-S] clusters and we here present an elegant evolutionarily conserved mechanism in BchL to protect its [4Fe-4S] cluster. We present a crystal structure of BchL in the nucleotide-free form with an ordered N-terminus that shields the [4Fe-4S] cluster at the docking interface between BchL and BchNB. Amino acid substitutions that perturb the shielding of the [4Fe-4S] cluster produce an unstable, but hyper-active enzyme complex, suggesting a role for the N-terminus in both auto-inhibition and enzyme stability. Upon ATP binding, a patch of amino acids, Asp-Phe-Asp (‘DFD patch’), situated at the mouth of the BchL ATP-binding pocket promotes inter-subunit cross stabilization of the two subunits and relieves the auto-inhibition by the N-terminus. A linked BchL dimer with one defective ATP-binding site does not support substrate reduction, illustrating that nucleotide binding to both subunits is a prerequisite for the inter-subunit cross stabilization. We propose that ATP-binding produces a conformational compaction of the BchL homodimer leading to a release of the flexible N-terminus from blocking the [4Fe-4S] cluster and promotes complex formation with BchNB to drive electron transfer. The auto-inhibitive feature and release mechanism appear unique to DPOR and is not found in the structurally similar nitrogenase.

scholarly journals Crizotinib acts as ABL1 inhibitor combining ATP-binding with allosteric inhibition and is active against native BCR-ABL1 and its resistance and compound mutants BCR-ABL1T315I and BCR-ABL1T315I-E255K

2021 ◽  
Author(s):  
Afsar Ali Mian ◽  
Isabella Haberbosch ◽  
Hazem Khamaisie ◽  
Abed Agbarya ◽  
Larissa Pietsch ◽  
...  
Keyword(s):  
Binding Site ◽  
Kinase Inhibitors ◽  
Therapeutic Potential ◽  
Lymphoblastic Leukemia ◽  
Philadelphia Chromosome ◽  
Binding Pocket ◽  
Atp Binding ◽  
Atp Binding Site ◽  
Lung Cancer Patients

AbstractResistance remains the major clinical challenge for the therapy of Philadelphia chromosome–positive (Ph+) leukemia. With the exception of ponatinib, all approved tyrosine kinase inhibitors (TKIs) are unable to inhibit the common “gatekeeper” mutation T315I. Here we investigated the therapeutic potential of crizotinib, a TKI approved for targeting ALK and ROS1 in non-small cell lung cancer patients, which inhibited also the ABL1 kinase in cell-free systems, for the treatment of advanced and therapy-resistant Ph+ leukemia. By inhibiting the BCR-ABL1 kinase, crizotinib efficiently suppressed growth of Ph+ cells without affecting growth of Ph− cells. It was also active in Ph+ patient-derived long-term cultures (PD-LTCs) independently of the responsiveness/resistance to other TKIs. The efficacy of crizotinib was confirmed in vivo in syngeneic mouse models of BCR-ABL1- or BCR-ABL1T315I-driven chronic myeloid leukemia–like disease and in BCR-ABL1-driven acute lymphoblastic leukemia (ALL). Although crizotinib binds to the ATP-binding site, it also allosterically affected the myristol binding pocket, the binding site of GNF2 and asciminib (former ABL001). Therefore, crizotinib has a seemingly unique double mechanism of action, on the ATP-binding site and on the myristoylation binding pocket. These findings strongly suggest the clinical evaluation of crizotinib for the treatment of advanced and therapy-resistant Ph+ leukemia.

scholarly journals Crystal structure of microtubule affinity-regulating kinase 4 catalytic domain in complex with a pyrazolopyrimidine inhibitor

2016 ◽  
Vol 72 (2) ◽  
pp. 129-134 ◽  
Author(s):  
John S. Sack ◽  
Mian Gao ◽  
Susan E. Kiefer ◽  
Joseph E. Myers ◽  
John A. Newitt ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Catalytic Domain ◽  
Neurofibrillary Tangle ◽  
Binding Pocket ◽  
Atp Binding ◽  
Activation Loop ◽  
Serine Threonine Kinase ◽  
Atp Binding Site ◽  
Threonine Kinase ◽  
Catalytic Loop

Microtubule-associated protein/microtubule affinity-regulating kinase 4 (MARK4) is a serine/threonine kinase involved in the phosphorylation of MAP proteins that regulate microtubule dynamics. Abnormal activity of MARK4 has been proposed to contribute to neurofibrillary tangle formation in Alzheimer's disease. The crystal structure of the catalytic and ubiquitin-associated domains of MARK4 with a potent pyrazolopyrimidine inhibitor has been determined to 2.8 Å resolution with anRworkof 22.8%. The overall structure of MARK4 is similar to those of the other known MARK isoforms. The inhibitor is located in the ATP-binding site, with the pyrazolopyrimidine group interacting with the inter-lobe hinge region while the aminocyclohexane moiety interacts with the catalytic loop and the DFG motif, forcing the activation loop out of the ATP-binding pocket.

Protein-Protein Interactions (PPIs) as an Alternative to Targeting the ATP Binding Site of Kinase

2016 ◽  
pp. 249-277
Author(s):  
Sailu Sarvagalla ◽  
Mohane Selvaraj Coumar
Keyword(s):  
Protein Interactions ◽  
In Silico ◽  
Rational Design ◽  
Kinase Activity ◽  
Hot Spot ◽  
Binding Pocket ◽  
Atp Binding ◽  
Protein Protein Interactions ◽  
Structure Based Drug Design ◽  
Atp Binding Site

Most of the developed kinase inhibitor drugs are ATP competitive and suffer from drawbacks such as off-target kinase activity, development of resistance due to mutation in the ATP binding pocket and unfavorable intellectual property situations. Besides the ATP binding pocket, protein kinases have binding sites that are involved in Protein-Protein Interactions (PPIs); these PPIs directly or indirectly regulate the protein kinase activity. Of recent, small molecule inhibitors of PPIs are emerging as an alternative to ATP competitive agents. Rational design of inhibitors for kinase PPIs could be carried out using molecular modeling techniques. In silico tools available for the prediction of hot spot residues and cavities at the PPI sites and the means to utilize this information for the identification of inhibitors are discussed. Moreover, in silico studies to target the Aurora B-INCENP PPI sites are discussed in context. Overall, this chapter provides detailed in silico strategies that are available to the researchers for carrying out structure-based drug design of PPI inhibitors.

Converting a weaker ATP-binding site inhibitor into a potent hetero-bivalent ligand by tethering to a unique peptide sequence derived from the same kinase

2018 ◽  
Vol 16 (35) ◽  
pp. 6443-6449 ◽  
Author(s):  
Samanth Reddy Kedika ◽  
D. Gomika Udugamasooriya
Keyword(s):  
Binding Site ◽  
Binding Pocket ◽  
Peptide Sequence ◽  
Atp Binding ◽  
Bivalent Ligand ◽  
Unique Peptide ◽  
Atp Binding Site

Our hetero-bivalent ligand targets a distance binding pocket to ATP-binding site using a peptide sequence already exists within same kinase.

scholarly journals Recent Advances in the Discovery of CK2 Allosteric Inhibitors: From Traditional Screening to Structure-Based Design

Molecules ◽  
2020 ◽  
Vol 25 (4) ◽  
pp. 870 ◽  
Author(s):  
Xiaolan Chen ◽  
Chunqiong Li ◽  
Dada Wang ◽  
Yu Chen ◽  
Na Zhang
Keyword(s):  
Protein Kinase Ck2 ◽  
Binding Pocket ◽  
Atp Binding ◽  
Binding Modes ◽  
Allosteric Site ◽  
Allosteric Inhibitors ◽  
Atp Binding Site ◽  
Recent Advances ◽  
Allosteric Sites ◽  
Kinase Ck2

Protein kinase (CK2) has emerged as an attractive cancer therapeutic target and recent efforts have been made to develop its inhibitors. However, the development of selective inhibitors remains challenging because of the highly conserved ATP-binding pocket (orthosteric site) of kinase family. As an alternative strategy, allosteric inhibitors, by targeting the much more diversified allosteric site relative to the conserved ATP-binding site, achieve better pharmacological advantages than orthosteric inhibitors. Traditional serendipitous screening and structure-based design are robust tools for the discovery of CK2 allosteric inhibitors. In this review, we summarize the recent advances in the identification of CK2 allosteric inhibitors. Firstly, we briefly present the CK2 allosteric sites. Then, the allosteric inhibitors targeting the well-elucidated allosteric sites (α/β interface, αD pocket and interface between the Glycine-rich loop and αC-helix) are highlighted in the discovery process and possible binding modes with the allosteric sites are described. This study is expected to provide valuable clues for the design of CK2 allosteric inhibitors.

scholarly journals Structural basis of rifampin inactivation by rifampin phosphotransferase

2016 ◽  
Vol 113 (14) ◽  
pp. 3803-3808 ◽  
Author(s):  
Xiaofeng Qi ◽  
Wei Lin ◽  
Miaolian Ma ◽  
Chengyuan Wang ◽  
Yang He ◽  
...  
Keyword(s):  
Molecular Mechanism ◽  
Conformational Changes ◽  
Bacterial Infections ◽  
Binding Pocket ◽  
Resistance Mechanisms ◽  
Binding Domain ◽  
Structural Basis ◽  
Atp Binding ◽  
Toggle Switch ◽  
The Cost

Rifampin (RIF) is a first-line drug used for the treatment of tuberculosis and other bacterial infections. Various RIF resistance mechanisms have been reported, and recently an RIF-inactivation enzyme, RIF phosphotransferase (RPH), was reported to phosphorylate RIF at its C21 hydroxyl at the cost of ATP. However, the underlying molecular mechanism remained unknown. Here, we solve the structures of RPH from Listeria monocytogenes (LmRPH) in different conformations. LmRPH comprises three domains: an ATP-binding domain (AD), an RIF-binding domain (RD), and a catalytic His-containing domain (HD). Structural analyses reveal that the C-terminal HD can swing between the AD and RD, like a toggle switch, to transfer phosphate. In addition to its catalytic role, the HD can bind to the AD and induce conformational changes that stabilize ATP binding, and the binding of the HD to the RD is required for the formation of the RIF-binding pocket. A line of hydrophobic residues forms the RIF-binding pocket and interacts with the 1-amino, 2-naphthol, 4-sulfonic acid and naphthol moieties of RIF. The R group of RIF points toward the outside of the pocket, explaining the low substrate selectivity of RPH. Four residues near the C21 hydroxyl of RIF, His825, Arg666, Lys670, and Gln337, were found to play essential roles in the phosphorylation of RIF; among these the His825 residue may function as the phosphate acceptor and donor. Our study reveals the molecular mechanism of RIF phosphorylation catalyzed by RPH and will guide the development of a new generation of rifamycins.

Sign in / Sign up

Export Citation Format

Share Document