The quiet renaissance of protein nuclear magnetic resonance

Abstract

From roughly 1985 through the start of the new millennium, the cutting edge of solution protein nuclear magnetic resonance (NMR) spectroscopy was to a significant extent driven by the aspiration to determine structures. Here we survey recent advances in protein NMR that herald a renaissance in which a number of its most important applications reflect the broad problem-solving capability displayed by this method during its classical era during the 1970s and early 1980s.

Figure 1

800 MHz ¹H-¹⁵N TROSY spectra of low concentrations of human arrestin-1 at 308 K in the absence and presence of bicelle-associated rhodopsin. (A) ¹H,¹⁵N-TROSY spectrum of 10 uM wild-type ²H,¹⁵N-labeled arrestin-1 in pH 6.5 buffer. (B) ¹H, ¹⁵N-TROSY spectra of 30 uM ²H, ¹⁵N-labeled F85A/F197A-arrestin-1 (black) in the presence of a saturating level (60 uM) of light-activated, phosphorylated rhodopsin P-Rh* in bicelles (red) at pH 6.5. Panel B is adapted from and used with permission of the publisher.

Figure 2

(A) Crystal structure of the inactive state of β2AR, showing the locations of the cysteines that were ¹⁹F labeled for studies of classical versus biased-agonism by the Stevens and Wüthrich labs. (B) ¹⁹F NMR spectra of C265- (left) and C327-¹⁹F-labeled (right) β2AR at 280°K in β-dodecylmaltoside micelles containing 17 mol% cholesterol hemisuccinate. Each spectrum has been deconvoluted into two spectral components, blue arising from the inactive state and red representing the activated state. It can be seen that the ratios of the two states (as judged by spectra from C265-label vs. C327-label) are, for most compounds, not equal. The simplest explanation for this observation is that there are actually two active states—one being the classical G-protein-coupling activated state, the other being the C-terminal phosphorylation-activated state that leads to binding of β-arrestin. (C) The series of compounds examined in this work classified according to their known pharmacological effects on β2AR. Inverse agonists preferentially stabilize the inactive receptor state. Neutral antagonists bind to the receptor without altering the basal inactive/active state ratio. Partial agonists result in sub-maximal conversion into the activated state, while full agonists result in maximal conversion. Carvedilol and isotherine are considered biased agonists in that they preferentially promote activation of complex formation with β-arrestin, although they differ with regards to the extent to which they also stimulate classic agonism. Each compound is color coded to indicate the helices of the receptor that each moiety interacts with (see color coding in panel A). Figure adapted from and used by permission of the publisher.

Figure 3

(A) Binding of cholesterol to the C99 domain of the amyloid precursor protein. Binding is believed to involve docking of the flat and rigid cholesterol to the flat surface provided by tandem GXXXG motifs on the surface of C99’s upper transmembrane helix. Formation of hydrogen bonds between the hydroxyl headgroup of cholesterol and C99 involves a conformational change (or conformational selection) centering on a flexible loop that connects a short surface-associated helix and C99’s transmembrane domain. (B) Distinct classes of interaction between two transmembrane proteins that can now be distinguished using NMR methods. Panel B from^, and used by permission of the publisher.

Figure 4

The NMR observed (red) and crystal structure back-calculated (black) PRE values for the maltose binding protein (MBP) in the holo (maltose-associated) (A) and apo (B) states. Red bars on the top of the plots indicate regions where signal intensities are broadened beyond detection as a result of relaxation enhancements. The holo-MBP PRE values show excellent agreement with the crystal structure indicating that the maltose-bound holo (closed) state protein is relatively rigid when complexed with maltose under both NMR and crystal conditions. The discrepancies in (B) between the observed and back-calculated PRE values for the apo state indicate a rapidly-exchanging mixture of a pair of structural states. These were determined to be the open state (similar to the apo crystal structure) and a 5% partially closed conformer. This latter structure is distinct from both the apo and holo state crystal structures but may represent the excited state conformer that initially binds maltose, which then induces a transition to the stable holo state. The inset in panel B is a surface representation of MBP with the green surface showing the conformational space explored by the paramagnetic nitroxide label and the red surface highlighting the regions where observed and back-calculated PREs don’t agree. The electrostatic surface of the open state of apo MBP (C) highlights the sugar binding pocket, with (D) illustrating the differences in the MBP-CTD between the partially closed apo-MBP (green cylinders) and closed holo-MBP states (red cylinders). Thus the data in this figure show the utility of the PRE to detect and probe minor protein populations that are challenging to observe with other techniques. In this case the minor population observed in the apo state is thought to be critical for ligand recognition and induced fit transition to the stable holo maltose-MBP conformation. This figure is a composite from those in and used by permission of the publisher.

Figure 5

Structure and dynamics of the 670-kDa α₇β₇β₇α₇ proteasome core particle probed by methyl-TROSY. (A) Cross-section view of the proteasome revealing its lumen. The residues in red were shown to undergo concerted motion and are located in the antechamber near the entrance to the catalytic chamber (where active site threonine residues are blue). The resonances from V14 (shown in yellow) were observed to be highly exchanged-broadened, reflecting the even more severe broadening of (invisible) resonances from the adjacent residues 1–12 as a result of msec timescale motion. (B) Cross-section highlighting residues that change methyl TROSY chemical shifts upon truncation of the first 12 residues of the α subunit (see scale and color coding at bottom). The largest changes are seen for sites located at the narrowest point of the substrate entrance channel (V129) and inside the antechamber. This suggests that the 12 N-terminal residues missing in the crystal structure populate states in which they are reversibly folded into the antechamber through the entrance to the channel, where they act as a gate. The location of residues 13–18 in the crystal structure are shown in green. (Figure from and used by permission of the publisher. Caption is also adapted from that some reference).

Figure 6

(A) Illustration of fragment-based drug design (FBDD) and both protein- and ligand-detected screening. The binding of small molecule fragments to a protein target can be detected by NMR even when affinity is low (100 μM–10 mM) High-affinity ligands can be created by linking together low affinity fragments that bind to adjacent sites. In the protein-detected mode, peaks from nuclei located at the binding interface shift when a candidate molecular fragment binds, an approach that has the advantage of suggesting the location of the binding site in the target. In the ligand-detected mode, small molecules that bind to a target protein are identified based on peak shifts or peak broadening/disappearance as a result of binding. (B) Chemical evolution of ABT-263.

Figure 7

Reaction cycle for dihydrofolate reductase. Highlighted in this figure are excited states confirmed by NMR to be dynamically sampled by each ground state complex along the reaction pathway. NADPH and NADP⁺ are shown in gold, while substrate, product, and analogs are shown in magenta. Note the approximate matches between the NMR-determined rates for conformational changes and the rates for the adjacent chemical/binding steps. From , used by permission of the publisher.

Figure 8

Comparison of the folding intermediate and natives states of mutant Fyn SH3 as determined by NMR. Sites for both native and the folding intermediate states are color-coded according to their predicated Zagg (Zagg > 1—orange to red—indicates significant propensity to aggregate). The high propensity of the β1 strand to aggregate is effectively blocked by the adjacent β5 strand in the folded native state, but is a source of peril for this protein in the intermediate state. From , used by permission of the publisher.

Figure 9

Dynamic complex of multiply-phosphorylated pSic1 with Cdc4. From , used by permission of the publisher.