A synergy of activity, stability, and inhibitor-interaction of HIV-1 protease mutants evolved under drug-pressure.

A clinically-relevant, drug-resistant mutant of HIV-1 protease (PR), termed Flap+(I54V) and containing L10I, G48V, I54V and V82A mutations, is known to produce significant changes in the entropy and enthalpy balance of drug-PR interactions, compared to wild-type PR. A similar mutant, Flap+(I54A) , which evolves from Flap+(I54V) and contains the single change at residue 54 relative to Flap+(I54V) , does not. Yet, how Flap+(I54A) behaves in solution is not known. To understand the molecular basis of V54A evolution, we compared nuclear magnetic resonance (NMR) spectroscopy, fluorescence spectroscopy, isothermal titration calorimetry, and enzymatic assay data from four PR proteins: PR (pWT), Flap+(I54V) , Flap+(I54A) , and Flap+(I54) , a control mutant that contains only L10I, G48V and V82A mutations. Our data consistently show that selection to the smaller side chain at residue 54, not only decreases inhibitor affinity, but also restores the catalytic activity.

An NMR strategy to detect conformational differences in a protein complexed with highly analogous inhibitors in solution.

This manuscript presents an NMR strategy to investigate conformational differences in protein-inhibitor complexes, when the inhibitors tightly bind to a protein at sub-nanomolar dissociation constants and are highly analogous to each other. Using HIV-1 protease (PR), we previously evaluated amide chemical shift differences, ΔCSPs, of PR bound to darunavir (DRV) compared to PR bound to several DRV analogue inhibitors, to investigate subtle but significant long-distance conformation changes caused by the inhibitor's chemical moiety variation [Khan, S. N., Persons, J. D. Paulsen, J. L., Guerrero, M., Schiffer, C. A., Kurt-Yilmaz, N., and Ishima, R., Biochemistry, (2018), 57, 1652-1662]. However, ΔCSPs are not ideal for investigating subtle PR-inhibitor interface differences because intrinsic differences in the electron shielding of the inhibitors affect protein ΔCSPs. NMR relaxation is also not suitable as it is not sensitive enough to detect small conformational differences in rigid regions among similar PR-inhibitor complexes. Thus, to gain insight into conformational differences at the inhibitor-protein interface, we recorded 15N-half filtered NOESY spectra of PR bound to two highly analogous inhibitors and assessed NOEs between PR amide protons and inhibitor protons, between PR amide protons and hydroxyl side chains, and between PR amide protons and water protons. We also verified the PR amide-water NOEs using 2D water-NOE/ROE experiments. Differences in water-amide proton NOE peaks, possibly due to amide-protein hydrogen bonds, were observed between subunit A and subunit B, and between the DRV-bound form and an analogous inhibitor-bound form, which may contribute to remote conformational changes.

Probing Structural Changes among Analogous Inhibitor-Bound Forms of HIV-1 Protease and a Drug-Resistant Mutant in Solution by Nuclear Magnetic Resonance.

In the era of state-of-the-art inhibitor design and high-resolution structural studies, detection of significant but small protein structural differences in the inhibitor-bound forms is critical to further developing the inhibitor. Here, we probed differences in HIV-1 protease (PR) conformation among darunavir and four analogous inhibitor-bound forms and compared them with a drug-resistant mutant using nuclear magnetic resonance chemical shifts. Changes in amide chemical shifts of wild-type (WT) PR among these inhibitor-bound forms, ΔCSP, were subtle but detectable and extended >10 Å from the inhibitor-binding site, asymmetrically between the two subunits of PR. Molecular dynamics simulations revealed differential local hydrogen bonding as the molecular basis of this remote asymmetric change. Inhibitor-bound forms of the drug-resistant mutant also showed a similar long-range ΔCSP pattern. Differences in ΔCSP values of the WT and the mutant (ΔΔCSPs) were observed at the inhibitor-binding site and in the surrounding region. Comparing chemical shift changes among highly analogous inhibitors and ΔΔCSPs effectively eliminated local environmental effects stemming from different chemical groups and enabled exploitation of these sensitive parameters to detect subtle protein conformational changes and to elucidate asymmetric and remote conformational effects upon inhibitor interaction.

Recommendations for Lengthening of Magnetically Controlled Growing Rods in Children With Pacemakers.

PURPOSE OF THE STUDY: Pacemakers are currently identified as a contraindication for the use of magnetic growth rods (MGRs). This arises from concern that magnetic fields generated by the MGR external remote controller (ERC) during lengthening procedures may induce pacemaker dysfunction. We investigated (1) whether MGR lengthening affects pacemaker function, and (2) if the magnetic field of a pacemaker affects MGR lengthening. METHODS: MGRs were tested in conjunction with an magnetic resonance imaging-compatible pacemaker, which was connected to a virtual patient under continuous cardiac monitoring. To determine whether pacemaker function was affected during MGR lengthening, the electrocardiogram trace was monitored for arrhythmias, whereas an ERC was applied to lengthen the MGRs at varying distances from the pacemaker. To investigate if MGR lengthening was affected by the presence of a pacemaker, at the start and end of the experiment, the ability of the rods to fully elongate and shorten was tested to check for conservation of function. RESULTS: When the pacemaker was in normal mode, <16 cm away from the activated ERC during MGR lengthening, pacemaker function was affected by the ERC's magnetic forces. At this distance, prophylactically switching the pacemaker to tonic mode before lengthening prevented occurrence of inappropriate pacing discharges. No deleterious effect of the pacemaker's magnetic field on the MGR lengthening mechanism was identified. CONCLUSIONS: Magnetic resonance imaging-compatible pacemakers appear safe for concomitant use with MGRs, provided a pacemaker technician prophylactically switches the pacemaker to tonic function before outpatient lengthening procedures. CLINICAL RELEVANCE: This experiment was designed to provide the first safety information on MGR lengthening in children with pacemakers. Although currently a rare clinical scenario, with increasing use of MGRs, this clinical scenario may arise more frequently in the future.

Distribution of Pico- and Nanosecond Motions in Disordered Proteins from Nuclear Spin Relaxation.

Intrinsically disordered proteins and intrinsically disordered regions (IDRs) are ubiquitous in the eukaryotic proteome. The description and understanding of their conformational properties require the development of new experimental, computational, and theoretical approaches. Here, we use nuclear spin relaxation to investigate the distribution of timescales of motions in an IDR from picoseconds to nanoseconds. Nitrogen-15 relaxation rates have been measured at five magnetic fields, ranging from 9.4 to 23.5 T (400-1000 MHz for protons). This exceptional wealth of data allowed us to map the spectral density function for the motions of backbone NH pairs in the partially disordered transcription factor Engrailed at 11 different frequencies. We introduce an approach called interpretation of motions by a projection onto an array of correlation times (IMPACT), which focuses on an array of six correlation times with intervals that are equidistant on a logarithmic scale between 21 ps and 21 ns. The distribution of motions in Engrailed varies smoothly along the protein sequence and is multimodal for most residues, with a prevalence of motions around 1 ns in the IDR. We show that IMPACT often provides better quantitative agreement with experimental data than conventional model-free or extended model-free analyses with two or three correlation times. We introduce a graphical representation that offers a convenient platform for a qualitative discussion of dynamics. Even when relaxation data are only acquired at three magnetic fields that are readily accessible, the IMPACT analysis gives a satisfactory characterization of spectral density functions, thus opening the way to a broad use of this approach.

Fractional enrichment of proteins using [2-(13)C]-glycerol as the carbon source facilitates measurement of excited state 13Cα chemical shifts with improved sensitivity.

A selective isotope labeling scheme based on the utilization of [2-(13)C]-glycerol as the carbon source during protein overexpression has been evaluated for the measurement of excited state (13)Cα chemical shifts using Carr-Purcell-Meiboom-Gill (CPMG) relaxation dispersion (RD) experiments. As expected, the fractional incorporation of label at the Cα positions is increased two-fold relative to labeling schemes based on [2-(13)C]-glucose, effectively doubling the sensitivity of NMR experiments. Applications to a binding reaction involving an SH3 domain from the protein Abp1p and a peptide from the protein Ark1p establish that accurate excited state (13)Cα chemical shifts can be obtained from RD experiments, with errors on the order of 0.06 ppm for exchange rates ranging from 100 to 1000 s(-1), despite the small fraction of (13)Cα-(13)Cß spin-pairs that are present for many residue types. The labeling approach described here should thus be attractive for studies of exchanging systems using (13)Cα spin probes.