Substrate Binding by γ-Secretase: Conformational Dynamics of the Enzyme Active Site and Substrate Recognition with an Example of the Amyloid Precursor Protein

Detailed understanding of how conformational dynamics orchestrates function in allosteric regulation of recognition and catalysis remains ambiguous. Here, we simulate CypA using multiple-microsecond-long atomistic molecular dynamics in explicit solvent and carry out NMR experiments. We analyze a large amount of time-dependent multidimensional data with a coarse-grained approach and map key dynamical features within individual macrostates by defining dynamics in terms of residue–residue contacts. The effects of substrate binding are observed to be largely sensed at a location over 15 Å from the active site, implying its importance in allostery. Using NMR experiments, we confirm that a dynamic cluster of residues in this distal region is directly coupled to the active site. Furthermore, the dynamical network of interresidue contacts is found to be coupled and temporally dispersed, ranging over 4 to 5 orders of magnitude. Finally, using network centrality measures we demonstrate the changes in the communication network, connectivity, and influence of CypA residues upon substrate binding, mutation, and during catalysis. We identify key residues that potentially act as a bottleneck in the communication flow through the distinct regions in CypA and, therefore, as targets for future mutational studies. Mapping these dynamical features and the coupling of dynamics to function has crucial ramifications in understanding allosteric regulation in enzymes and proteins, in general.

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Molecular determinant of substrate binding and specificity of cytochrome P450 2J2

Scientific Reports ◽

10.1038/s41598-020-79284-0 ◽

2020 ◽

Vol 10 (1) ◽

Author(s):

Liang Xu ◽

Liao Y. Chen

Keyword(s):

Arachidonic Acid ◽

Cytochrome P450 ◽

Active Site ◽

Structural Model ◽

Substrate Binding ◽

Conformational Dynamics ◽

Structural Basis ◽

Molecular Determinant ◽

Dynamics Simulations

AbstractCytochrome P450 2J2 (CYP2J2) is responsible for the epoxidation of endogenous arachidonic acid, and is involved in the metabolism of exogenous drugs. To date, no crystal structure of CYP2J2 is available, and the proposed structural basis for the substrate recognition and specificity in CYP2J2 varies with the structural models developed using different computational protocols. In this study, we developed a new structural model of CYP2J2, and explored its sensitivity to substrate binding by molecular dynamics simulations of the interactions with chemically similar fluorescent probes. Our results showed that the induced-fit binding of these probes led to the preferred active poses ready for the catalysis by CYP2J2. Divergent conformational dynamics of CYP2J2 due to the binding of each probe were observed. However, a stable hydrophobic clamp composed of residues I127, F310, A311, V380, and I487 was identified to restrict any substrate access to the active site of CYP2J2. Molecular docking of a series of compounds including amiodarone, astemizole, danazol, ebastine, ketoconazole, terfenadine, terfenadone, and arachidonic acid to CYP2J2 confirmed the role of those residues in determining substrate binding and specificity of CYP2J2. In addition to the flexibility of CYP2J2, the present work also identified other factors such as electrostatic potential in the vicinity of the active site, and substrate strain energy and property that have implications for the interpretation of CYP2J2 metabolism.

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Recognition of the amyloid precursor protein by human γ-secretase

Science ◽

10.1126/science.aaw0930 ◽

2019 ◽

Vol 363 (6428) ◽

pp. eaaw0930 ◽

Cited By ~ 80

Author(s):

Rui Zhou ◽

Guanghui Yang ◽

Xuefei Guo ◽

Qiang Zhou ◽

Jianlin Lei ◽

...

Keyword(s):

Alzheimer’S Disease ◽

Alzheimer's Disease ◽

Amyloid Precursor Protein ◽

Atomic Structure ◽

Substrate Binding ◽

Peptide Bond ◽

Catalytic Subunit ◽

Precursor Protein ◽

Β Sheet ◽

Specific Inhibitors

Cleavage of amyloid precursor protein (APP) by the intramembrane protease γ-secretase is linked to Alzheimer’s disease (AD). We report an atomic structure of human γ-secretase in complex with a transmembrane (TM) APP fragment at 2.6-angstrom resolution. The TM helix of APP closely interacts with five surrounding TMs of PS1 (the catalytic subunit of γ-secretase). A hybrid β sheet, which is formed by a β strand from APP and two β strands from PS1, guides γ-secretase to the scissile peptide bond of APP between its TM and β strand. Residues at the interface between PS1 and APP are heavily targeted by recurring mutations from AD patients. This structure, together with that of γ-secretase bound to Notch, reveal contrasting features of substrate binding, which may be applied toward the design of substrate-specific inhibitors.

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Strain energy in enzyme–substrate binding: An energetic insight into the flexibility versus rigidity of enzyme active site

Computational and Theoretical Chemistry ◽

10.1016/j.comptc.2012.06.017 ◽

2012 ◽

Vol 995 ◽

pp. 17-23 ◽

Cited By ~ 19

Author(s):

Xinchun Guo ◽

Deyong He ◽

Ling Huang ◽

Limin Liu ◽

Lijun Liu ◽

...

Keyword(s):

Active Site ◽

Strain Energy ◽

Substrate Binding ◽

Enzyme Active Site ◽

Enzyme Substrate ◽

Insight Into

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Substrate binding interferes with active site conformational dynamics in endoglucanase Cel5A from Thermobifida fusca

Biochemical and Biophysical Research Communications ◽

10.1016/j.bbrc.2017.07.086 ◽

2017 ◽

Vol 491 (1) ◽

pp. 236-240 ◽

Cited By ~ 1

Author(s):

Xukai Jiang ◽

Yuying Wang ◽

Limei Xu ◽

Guanjun Chen ◽

Lushan Wang

Keyword(s):

Active Site ◽

Substrate Binding ◽

Conformational Dynamics ◽

Thermobifida Fusca

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SUBSTRATE BINDING INHIBITS γ-SECRETASE CLEAVAGE OF AMYLOID PRECURSOR PROTEIN

Alzheimer s & Dementia ◽

10.1016/j.jalz.2019.09.052 ◽

2019 ◽

Vol 15 (7) ◽

pp. P1595

Author(s):

Jing Zhao ◽

Yuanyuan Xiao ◽

Xinyue Liu ◽

Soohyun Kim ◽

Xianzhong Wu ◽

...

Keyword(s):

Amyloid Precursor Protein ◽

Substrate Binding ◽

Precursor Protein

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Hinge-shift mechanism as a protein design principle for the evolution of β-lactamases from substrate promiscuity to specificity

Nature Communications ◽

10.1038/s41467-021-22089-0 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Tushar Modi ◽

Valeria A. Risso ◽

Sergio Martinez-Rodriguez ◽

Jose A. Gavira ◽

Mubark D. Mebrat ◽

...

Keyword(s):

Protein Design ◽

Active Site ◽

Design Principle ◽

Conformational Dynamics ◽

Evolutionary Process ◽

Computational Approach ◽

Enzyme Specificity ◽

Enzyme Active Site ◽

Sequence Reconstruction ◽

Substrate Promiscuity

AbstractTEM-1 β-lactamase degrades β-lactam antibiotics with a strong preference for penicillins. Sequence reconstruction studies indicate that it evolved from ancestral enzymes that degraded a variety of β-lactam antibiotics with moderate efficiency. This generalist to specialist conversion involved more than 100 mutational changes, but conserved fold and catalytic residues, suggesting a role for dynamics in enzyme evolution. Here, we develop a conformational dynamics computational approach to rationally mold a protein flexibility profile on the basis of a hinge-shift mechanism. By deliberately weighting and altering the conformational dynamics of a putative Precambrian β-lactamase, we engineer enzyme specificity that mimics the modern TEM-1 β-lactamase with only 21 amino acid replacements. Our conformational dynamics design thus re-enacts the evolutionary process and provides a rational allosteric approach for manipulating function while conserving the enzyme active site.

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Memapsin 2, a drug target for Alzheimer's disease

Biochemical Society Symposium ◽

10.1042/bss0700213 ◽

2003 ◽

Vol 70 ◽

pp. 213-220 ◽

Cited By ~ 1

Author(s):

Gerald Koelsch ◽

Robert T. Turner ◽

Lin Hong ◽

Arun K. Ghosh ◽

Jordan Tang

Keyword(s):

Crystal Structure ◽

Alzheimer’S Disease ◽

Alzheimer's Disease ◽

Transition State ◽

Amyloid Precursor Protein ◽

Therapeutic Target ◽

Drug Target ◽

Aspartic Protease ◽

Precursor Protein ◽

Memapsin 2

Mempasin 2, a ϐ-secretase, is the membrane-anchored aspartic protease that initiates the cleavage of amyloid precursor protein leading to the production of ϐ-amyloid and the onset of Alzheimer's disease. Thus memapsin 2 is a major therapeutic target for the development of inhibitor drugs for the disease. Many biochemical tools, such as the specificity and crystal structure, have been established and have led to the design of potent and relatively small transition-state inhibitors. Although developing a clinically viable mempasin 2 inhibitor remains challenging, progress to date renders hope that memapsin 2 inhibitors may ultimately be useful for therapeutic reduction of ϐ-amyloid.

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