Rieko Ishima

Protein dynamics from NMR

This review surveys recent investigations of conformational fluctuations of proteins in solution using NMR techniques. Advances in experimental methods have provided more accurate means of characterizing fast and slow internal motions as well as overall diffusion. The information obtained from NMR dynamics experiments provides insights into specific structural changes or configurational energetics associated with function. A variety of applications illustrate that studies of protein dynamics provide insights into protein–protein interactions, target recognition, ligand binding, and enzyme function.

Solution structure of the homodimeric core domain of Escherichia coli histidine kinase EnvZ

Escherichia coli osmosensor EnvZ is a protein histidine kinase that plays a central role in osmoregulation, a cellular adaptation process involving the His-Asp phosphorelay signal transduction system. Dimerization of the transmembrane protein is essential for its autophosphorylation and phosphorelay signal transduction functions. Here we present the NMR-derived structure of the homodimeric core domain (residues 223–289) of EnvZ that includes His 243, the site of autophosphorylation and phosphate transfer reactions. The structure comprises a four-helix bundle formed by two identical helix-turn-helix subunits, revealing the molecular assembly of two active sites within the dimeric kinase.

Flap opening and dimer-interface flexibility in the free and inhibitor-bound HIV protease, and their implications for function

BACKGROUND (1)H and (15)N transverse relaxation measurements on perdeuterated proteins are ideally suited for detecting backbone conformational fluctuations on the millisecond-microsecond timescale. The identification of conformational exchange on this timescale by measuring the relaxation of both (1)H and (15)N holds great promise for the elucidation of functionally relevant conformational changes in proteins. RESULTS We measured the transverse (1)H and (15)N relaxation rates of backbone amides of HIV-1 protease in its free and inhibitor-bound forms. An analysis of these rates, obtained as a function of the effective rotating frame field, provided information about the timescale of structural fluctuations in several regions of the protein. The flaps that cover the active site of the inhibitor-bound protein undergo significant changes of backbone (φ,psi) angles, on the 100 micros timescale, in the free protein. In addition, the intermonomer beta-sheet interface of the bound form, which from protease structure studies appears to be rigid, was found to fluctuate on the millisecond timescale. CONCLUSIONS We present a working model of the flap-opening mechanism in free HIV-1 protease which involves a transition from a semi-open to an open conformation that is facilitated by interaction of the Phe53 ring with the substrate. We also identify a surprising fluctuation of the beta-sheet intermonomer interface that suggests a structural requirement for maturation of the protease. Thus, slow conformational fluctuations identified by (1)H and (15)N transverse relaxation measurements can be related to the biological functions of proteins.

Rapid structural fluctuations of the free HIV protease flaps in solution: relationship to crystal structures and comparison with predictions of dynamics calculations.

Crystal structures have shown that the HIV‐1 protease flaps, domains that control access to the active site, are closed when the active site is occupied by a ligand. Although flap structures ranging from closed to semi‐open are observed in the free protease, crystal structures reveal that even the semi‐open flaps block access to the active site, indicating that the flaps are mobile in solution. The goals of this paper are to characterize the secondary structure and fast (sub‐ns) dynamics of the flaps of the free protease in solution, to relate these results to X‐ray structures and to compare them with predictions of dynamics calculations. To this end we have obtained nearly complete backbone and many sidechain signal assignments of a fully active free‐protease construct that is stabilized against autoproteolysis by three point mutations. The secondary structure of this protein was characterized using the chemical shift index, measurements of 3hJNC′ couplings across hydrogen bonds, and NOESY connectivities. Analysis of these measurements indicates that the protease secondary structure becomes irregular near the flap tips, residues 49–53. Model‐free analysis of 15N relaxation parameters, T1, T2 (T1ρ) and 15N‐{1H} NOE, shows that residues in the flap tips are flexible on the sub‐ns time scale, in contrast with previous observations on the inhibitor‐bound protease. These results are compared with theoretical predictions of flap dynamics and the possible biological significance of the sub‐ns time scale dynamics of the flap tips is discussed.

Extending the range of amide proton relaxation dispersion experiments in proteins using a constant-time relaxation-compensated CPMG approach

Relaxation compensated constant-time Carr–Purcell–Meiboom–Gill relaxation dispersion experiments for amide protons are presented that detect μs-ms time-scale dynamics of protein backbone amide sites. Because of their ten-fold larger magnetogyric ratio, much shorter 180° pulses can be applied to 1H than to 15N spins; therefore, off-resonance effects are reduced and a wider range of effective rf fields can often be used in the case of 1H experiments. Applications to [1H-15N]-ubiquitin and [1H-15N]-perdeuterated HIV-1 protease are discussed. In the case of ubiquitin, we present a pulse sequence that reduces artifacts that arise from homonuclear 3J(HN-Hα) coupling. In the case of the protease, we show that relaxation dispersion of both 1H and 15N spins provides a more comprehensive picture of slow backbone dynamics than does the relaxation dispersion of either spin alone. We also compare the relative merits of 1H versus 15N transverse relaxation measurements and note the benefits of using a perdeuterated protein to measure the relaxation dispersion of both spin types.

HIV-1 protease: structure, dynamics, and inhibition.

Publisher Summary This chapter focuses on some new and evolving areas of protease research. The human immunodeficiency virus type 1 (HIV‐1) protease is synthesized as part of a large Gag‐Pol precursor protein. The probing structure and dynamics of the free and inhibited mature protease dimer by nuclear magnetic resonance (NMR) to gain insights into specific regions of the dimer and their relationship to function is discussed in the chapter. It discusses the crystal structures at atomic resolutions of wild‐type and drug‐resistant mutant proteases in complex with substrate analogues and the comparison of structures using previous and new generations of active site inhibitors to understand the molecular mechanisms of drug resistance. The mutational and structural studies aimed at characterizing the monomer of the mature protease and its precursor is described in the chapter. The latter studies complement and form a basis for ongoing and future studies aimed at targeting protease dimerization, thus, extending the target area of current inhibitors, all of which bind across the active site formed by both sub units in the active dimer.

Engineered Human Antibody Constant Domains with Increased Stability

The immunoglobulin (Ig) constant CH2 domain is critical for antibody effector functions. Isolated CH2 domains are promising as scaffolds for construction of libraries containing diverse binders that could also confer some effector functions. However, previous work has shown that an isolated murine CH2 domain is relatively unstable to thermally induced unfolding. To explore unfolding mechanisms of isolated human CH2 and increase its stability γ1 CH2 was cloned and a panel of cysteine mutants was constructed. Human γ1 CH2 unfolded at a higher temperature (Tm = 54.1 °C, as measured by circular dichroism) than that previously reported for a mouse CH2 (41 °C). One mutant (m01) was remarkably stable (Tm = 73.8 °C). Similar results were obtained by differential scanning calorimetry. This mutant was also significantly more stable than the wild-type CH2 against urea induced unfolding (50% unfolding at urea concentration of 6.8 m versus 4.2 m). The m01 was highly soluble and monomeric. The existence of the second disulfide bond in m01 and its correct position were demonstrated by mass spectrometry and nuclear magnetic resonance spectroscopy, respectively. The loops were on average more flexible than the framework in both CH2 and m01, and the overall secondary structure was not affected by the additional disulfide bond. These data suggest that a human CH2 domain is relatively stable to unfolding at physiological temperature, and that both CH2 and the highly stable mutant m01 are promising new scaffolds for the development of therapeutics against human diseases.

A solution NMR study of the binding kinetics and the internal dynamics of an HIV‐1 protease‐substrate complex

NMR studies of the binding of a substrate to an inactive HIV‐1 protease construct, containing an active site mutation PRD25N, are reported. Substrate titration measurements monitored by HSQC spectra and a 15N‐edited NOESY experiment show that the chromogenic substrate analog of the capsid/p2 cleavage site binds to PRD25N with an equilibrium dissociation constant, KD, of 0.27 ± 0.05 mM, and upper limits of the association and dissociation rate constants, 2×104 M−1s−1 and 10 s−1, respectively, at 20°C, pH 5.8. This association rate constant is not in the diffusion limit, suggesting that association is controlled by a rare event, such as opening of the protease flaps. Analysis of 15N relaxation experiments reveals a slight reduction of S2 values in the flap region, indicating a small increase in the amplitude of internal motion on the sub‐nsec timescale. In addition, several residues in the flap region are mobile on the conformational exchange timescale, msec–μsec. Flap dynamics of the protease‐substrate complex are compared with those of protease‐inhibitor complexes, and the implications of these results for substrate‐binding models are discussed.

Solution structure of the mature HIV-1 protease monomer: Insight into the tertiary fold and stability of a precursor

We present the first solution structure of the HIV-1 protease monomer spanning the region Phe1–Ala95 (PR1–95). Except for the terminal regions (residues 1–10 and 91–95) that are disordered, the tertiary fold of the remainder of the protease is essentially identical to that of the individual subunit of the dimer. In the monomer, the side chains of buried residues stabilizing the active site interface in the dimer, such as Asp25, Asp29, and Arg87, are now exposed to solvent. The flap dynamics in the monomer are similar to that of the free protease dimer. We also show that the protease domain of an optimized precursor flanked by 56 amino acids of the N-terminal transframe region is predominantly monomeric, exhibiting a tertiary fold that is quite similar to that of PR1–95 structure. This explains the very low catalytic activity observed for the protease prior to its maturation at its N terminus as compared with the mature protease, which is an active stable dimer under identical conditions. Adding as few as 2 amino acids to the N terminus of the mature protease significantly increases its dissociation into monomers. Knowledge of the protease monomer structure and critical features of its dimerization may aid in the screening and design of compounds that target the protease prior to its maturation from the Gag-Pol precursor.

Effect of the Active Site D25N Mutation on the Structure, Stability, and Ligand Binding of the Mature HIV-1 Protease

All aspartic proteases, including retroviral proteases, share the triplet DTG critical for the active site geometry and catalytic function. These residues interact closely in the active, dimeric structure of HIV-1 protease (PR). We have systematically assessed the effect of the D25N mutation on the structure and stability of the mature PR monomer and dimer. The D25N mutation (PRD25N) increases the equilibrium dimer dissociation constant by a factor >100-fold (1.3 ± 0.09 μm) relative to PR. In the absence of inhibitor, NMR studies reveal clear structural differences between PR and PRD25N in the relatively mobile P1 loop (residues 79-83) and flap regions, and differential scanning calorimetric analyses show that the mutation lowers the stabilities of both the monomer and dimer folds by 5 and 7.3 °C, respectively. Only minimal differences are observed in high resolution crystal structures of PRD25N complexed to darunavir (DRV), a potent clinical inhibitor, or a non-hydrolyzable substrate analogue, Ac-Thr-Ile-Nle-r-Nle-Gln-Arg-NH2 (RPB), as compared with PR·DRV and PR·RPB complexes. Although complexation with RPB stabilizes both dimers, the effect on their Tm is smaller for PRD25N (6.2 °C) than for PR (8.7 °C). The Tm of PRD25N·DRV increases by only 3 °C relative to free PRD25N, as compared with a 22 °C increase for PR·DRV, and the mutation increases the ligand dissociation constant of PRD25N·DRV by a factor of ∼106 relative to PR·DRV. These results suggest that interactions mediated by the catalytic Asp residues make a major contribution to the tight binding of DRV to PR.