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

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.

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

Journal of Biological Chemistry ◽

10.1074/jbc.ra120.016278 ◽

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.

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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

Annals of Hematology ◽

10.1007/s00277-020-04357-z ◽

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.

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

Acta Crystallographica Section F Structural Biology Communications ◽

10.1107/s2053230x15024747 ◽

2016 ◽

Vol 72 (2) ◽

pp. 129-134 ◽

Cited By ~ 22

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.

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

Advances in Medical Technologies and Clinical Practice - Applied Case Studies and Solutions in Molecular Docking-Based Drug Design ◽

10.4018/978-1-5225-0362-0.ch010 ◽

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.

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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

Organic & Biomolecular Chemistry ◽

10.1039/c8ob01406j ◽

2018 ◽

Vol 16 (35) ◽

pp. 6443-6449 ◽

Cited By ~ 2

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.

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

Molecules ◽

10.3390/molecules25040870 ◽

2020 ◽

Vol 25 (4) ◽

pp. 870 ◽

Cited By ~ 1

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.

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

Pharmaceutical Sciences ◽

10.4018/978-1-5225-1762-7.ch043 ◽

2017 ◽

pp. 1115-1143

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

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New Mechanistic Insight on the PIM-1 Kinase Inhibitor AZD1208 Using Multidrug Resistant Human Erythroleukemia Cell Lines and Molecular Docking Simulations

Current Topics in Medicinal Chemistry ◽

10.2174/1568026619666190509121606 ◽

2019 ◽

Vol 19 (11) ◽

pp. 914-926 ◽

Cited By ~ 2

Author(s):

Maiara Bernardes Marques ◽

Michael González-Durruthy ◽

Bruna Félix da Silva Nornberg ◽

Bruno Rodrigues Oliveira ◽

Daniela Volcan Almeida ◽

...

Keyword(s):

Molecular Docking ◽

Binding Site ◽

Cell Lines ◽

Chemotherapeutic Agents ◽

Atp Binding ◽

Human Leukemia ◽

Atp Binding Site ◽

Mechanistic Insight ◽

Multiple Drugs

Background:PIM-1 is a kinase which has been related to the oncogenic processes like cell survival, proliferation, and multidrug resistance (MDR). This kinase is known for its ability to phosphorylate the main extrusion pump (ABCB1) related to the MDR phenotype.Objective:In the present work, we tested a new mechanistic insight on the AZD1208 (PIM-1 specific inhibitor) under interaction with chemotherapy agents such as Daunorubicin (DNR) and Vincristine (VCR).Materials and Methods:In order to verify a potential cytotoxic effect based on pharmacological synergism, two MDR cell lines were used: Lucena (resistant to VCR) and FEPS (resistant to DNR), both derived from the K562 non-MDR cell line, by MTT analyses. The activity of Pgp was ascertained by measuring accumulation and the directional flux of Rh123. Furthermore, we performed a molecular docking simulation to delve into the molecular mechanism of PIM-1 alone, and combined with chemotherapeutic agents (VCR and DNR).Results:Our in vitro results have shown that AZD1208 alone decreases cell viability of MDR cells. However, co-exposure of AZD1208 and DNR or VCR reverses this effect. When we analyzed the ABCB1 activity AZD1208 alone was not able to affect the pump extrusion. Differently, co-exposure of AZD1208 and DNR or VCR impaired ABCB1 activity, which could be explained by compensatory expression of abcb1 or other extrusion pumps not analyzed here. Docking analysis showed that AZD1208 is capable of performing hydrophobic interactions with PIM-1 ATP- binding-site residues with stronger interaction-based negative free energy (FEB, kcal/mol) than the ATP itself, mimicking an ATP-competitive inhibitory pattern of interaction. On the same way, VCR and DNR may theoretically interact at the same biophysical environment of AZD1208 and also compete with ATP by the PIM-1 active site. These evidences suggest that AZD1208 may induce pharmacodynamic interaction with VCR and DNR, weakening its cytotoxic potential in the ATP-binding site from PIM-1 observed in the in vitro experiments.Conclusion:Finally, the current results could have a pre-clinical relevance potential in the rational polypharmacology strategies to prevent multiple-drugs resistance in human leukemia cancer therapy.

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