Pulsed EPR characterization of HIV-1 protease conformational sampling and inhibitor-induced population shifts

Liu, Zhanglong; Casey, Thomas M.; Blackburn, Mandy E.; Huang, Xi Ping; Pham, Linh; Vera, Ian Mitchelle S. de; Carter, Jeffrey D.; Kear-Scott, Jamie L.; Veloro, Angelo M.; Galiano, Luis; Fanucci, Gail E.

doi:10.1039/c5cp04556h

Cited by 25 publications

(33 citation statements)

References 109 publications

(340 reference statements)

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“…The overall structure of the HIV-1 protease is rigidified upon Ab/RIT binding or upon mutation. HIV-1 protease is widely studied by techniques including nuclear magnetic resonance (NMR) [36][37][38][39][40] , X-ray crystallography [41][42][43] , electron paramagnetic resonance (EPR) 17,20 , and molecular dynamics simulations 23,35,44,45 . NMR experiments suggested large scale flap dynamics of unliganded form of HIV-1 protease.…”

Section: Discussionmentioning

confidence: 99%

“…We also found that CDRs of the antibody are highly flexible and they adjust themselves to accommodate different conformations of the same epitope sequence (P36-45) in peptide and protein antigens. Thus, our study explained the cross-reactivity of P36-45 peptide and protease with antibody 12 .There is a large diversity in the flap conformations in the unbound state, fluctuating between the closed, semi-open, and wide-open conformations [13][14][15][16][17] . In the closed/semi-open conformation, the catalytic site is covered by two flaps, and thus restricts the entry of most of the ligands.…”

mentioning

confidence: 84%

“…There is a large diversity in the flap conformations in the unbound state, fluctuating between the closed, semi-open, and wide-open conformations [13][14][15][16][17] . In the closed/semi-open conformation, the catalytic site is covered by two flaps, and thus restricts the entry of most of the ligands.…”

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confidence: 99%

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Inhibition of the activity of HIV-1 protease through antibody binding and mutations probed by molecular dynamics simulations

Badaya

Sasidhar

2020

Sci Rep

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HIV-1 protease is an essential enzyme in the life cycle of the HIV-1 virus. The conformational dynamics of the flap region of the protease is critical for the ligand binding mechanism, as well as for the catalytic activity. The monoclonal antibody F11.2.32 raised against HIV-1 protease inhibits its activity on binding. We have studied the conformational dynamics of protease in its free, inhibitor ritonavir and antibody bound forms using molecular dynamics simulations. We find that upon Ab binding to the epitope region (residues 36-46) of protease, the overall flexibility of the protease is decreased including the flap region and the active site, which is similar to the decrease in flexibility observed by inhibitor binding to the protease. This suggests an allosteric mechanism to inhibit protease activity. Further, the protease mutants G40E and G40R are known to have decreased activity and were also subjected to MD simulations. We find that the loss of flexibility in the mutants is similar to that observed in the protease bound to the Ab/inhibitor. these insights highlight the role played by dynamics in the function of the protease and how control of flexibility through Ab binding and site specific mutations can inhibit protease activity.The HIV-1 protease belongs to the family of aspartyl protease. It cleaves the newly synthesized polyproteins, which is the vital step to create the mature protein components of an infectious HIV-1 virus 1 . Thus, HIV-1 protease is an essential enzyme in the life-cycle of the HIV-1 virus and a potential target for the structure-based drug design. There are several commercial drugs available in the market against the HIV-1 protease. The success rate of these drugs is low due to the occurrence of drug-resistant mutations in the HIV-1 protease 2 . For example, the thermodynamic integration (TI) and MD simulation studies by Chen and his group suggested that there is a change in the shape and conformation of the binding pocket upon certain drug-resistant mutations and this consequently reduces the binding affinity of inhibitor 3 . Also, conventional method of drug delivery targets active sites and many enzymes with related function may have very similar active sites. This may cause adverse side-effects. Therefore, there is a requirement to generate new generation drugs that can function away from active-site and these are allosteric drugs 4-7 . To identify a new target on HIV-1 protease, there is a need to understand the complete structure and dynamics of HIV-1 protease to inhibits its enzymatic activity. The HIV-1 protease is a homodimeric enzyme with each monomer comprising of 99 amino-acid residues 8 . The active site (residues Asp25, Thr26 and Gly27 from both chains A and B) of the protease is covered by two flaps (residues 43-58) from each chain 9,10 . F11.2.32 is a monoclonal antibody (mAb) raised against the HIV-1 protease. The peptide P36-46 ( 36 MNLPGRWKPKM 46 ) corresponding to the epitope/elbow region of the protease binds to the complementarity determining regions (CDRs) of ...

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Section: Discussionmentioning

confidence: 99%

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confidence: 84%

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Inhibition of the activity of HIV-1 protease through antibody binding and mutations probed by molecular dynamics simulations

Badaya

Sasidhar

2020

Sci Rep

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“…The development of selective and non-perturbing molecular probes is vital for understanding the complex interactions of biological systems with biomolecules on surfaces.N itroxide radical spin labels for electron paramagnetic resonance (EPR) spectroscopy are an effective way to monitor protein dynamics. [1][2][3] By using nitroxide spin labels,binding of various enzymes, [4] lectins, [5] RNA, [6] and other bio-macromolecules may be observed and quantified with EPR, offering opportunities to understand interfacial recognition at surfaces, including how receptor density on cell surfaces affects multivalent interactions with proteins.…”

mentioning

confidence: 99%

“…[1][2][3] By using nitroxide spin labels,binding of various enzymes, [4] lectins, [5] RNA, [6] and other bio-macromolecules may be observed and quantified with EPR, offering opportunities to understand interfacial recognition at surfaces, including how receptor density on cell surfaces affects multivalent interactions with proteins.…”

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A Bifunctional Spin Label for Ligand Recognition on Surfaces

et al. 2017

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In situ monitoring of biomolecular recognition, especially at surfaces,s till presents as ignificant technical challenge.E lectron paramagnetic resonance (EPR) of biomolecules spin-labeled with nitroxides can offer uniquely sensitive and selective insights into these processes,b ut new spin-labeling strategies are needed. The synthesis and study of ab romoacrylaldehyde spin label (BASL), which features two attachment points with orthogonal reactivity is reported. The first examples of mannose and biotin ligands coupled to aqueous carboxy-functionalized gold nanoparticles through as pin label are presented. EPR spectra were obtained for the spin-labeled ligands both free in solution and attached to nanoparticles.T he labels were recognized by the mannosebinding lectin, Con A, and the biotin-binding protein avidinperoxidase.B inding gave quantifiable changes in the EPR spectra from which binding profiles could be obtained that reflect the strength of binding in eachc ase.

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Double Histidine Based EPR Measurements at Physiological Temperatures Permit Site‐Specific Elucidation of Hidden Dynamics in Enzymes

et al. 2020

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Protein dynamics is at the heart of all cellular processes. Here, we utilize the dHis-Cu II NTA label to obtain site-specific information on dynamics for both an a-helix and b-sheet site of GB1, the immunoglobulin binding domain of protein G. Spectral features found in our CW-EPR measurements were consistent with the overall rigid nature of GB1 and with predictions from molecular dynamics simulations. Using this information, we show the potential of this approach to elucidate the role of dynamics in substrate binding of a functionally necessary a-helix in human glutathione transferase A1-1 (hGSTA1-1). We observe two dynamical modes for the helix. The addition of the inhibitor GS-Met and GS-Hex resulted in hGSTA1-1 to favor the more rigid active state conformation, while the faster mode potentially aids the search for substrates. Together the results illustrate the remarkable potential of the dHis-based labelling approach to measure sitespecific dynamics using room temperature lineshape analysis.

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Pulsed EPR characterization of HIV-1 protease conformational sampling and inhibitor-induced population shifts

Cited by 25 publications

References 109 publications

Inhibition of the activity of HIV-1 protease through antibody binding and mutations probed by molecular dynamics simulations

Inhibition of the activity of HIV-1 protease through antibody binding and mutations probed by molecular dynamics simulations

A Bifunctional Spin Label for Ligand Recognition on Surfaces

Double Histidine Based EPR Measurements at Physiological Temperatures Permit Site‐Specific Elucidation of Hidden Dynamics in Enzymes

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