A combination of NMR techniques is able to explore the structure of short-lived protein conformations.
Proteins can fold into many different conformations, and the conformation with the lowest energy is the most stable (Frauenfelder et al., 1991). Two techniques—X-ray crystallography and NMR spectroscopy—are routinely used to work out the structure of the protein in its ground state with atomic resolution. However, a given protein also spends time in other (higher-energy) states, and it has become clear over the past decade that these other states are involved in various biological processes, including protein folding, enzyme catalysis, protein-ligand interactions and protein allostery (Palmer, 2004; Sekhar and Kay, 2013; Tzeng and Kalodimos, 2013). However, as few of the proteins in a sample will be in any of these higher-energy states, most biophysical techniques are not able to detect them.
NMR spin relaxation is a powerful technique that can probe how proteins move over a wide range of timescales, from picoseconds to hours, with atomic resolution (Kay, 2011). Now, in eLife, Lewis Kay of the University of Toronto and co-workers—including Ashok Sekhar as first author—have used two complementary NMR spin relaxation methods to explore the different conformations of an enzyme that has been linked to amyotrophic lateral sclerosis (ALS), a devastating neurodegenerative disease (Sekhar et al., 2015).
Mutations in an enzyme called SOD1 have been identified as the main cause of the inherited form of ALS (Rosen et al., 1993). However, little is known about the cause of sporadic ALS, which has the same symptoms but appears to occur randomly throughout the population. The detailed molecular mechanisms of ALS remain to be clarified, although the inherited and sporadic forms of the disease are thought to share a common pathway. The detection of insoluble SOD1 in ALS patients suggests that mutations and modifications could lead to conformational changes in SOD1 that, in turn, increase the chances that it will misfold and form insoluble aggregates (Bosco et al., 2010).
The SOD1 enzyme can take on many different structural forms. The active form of the enzyme, known as Cu2Zn2SOD1S-S, exists as a dimer made up of two identical subunits. Each subunit is a β-barrel with eight strands, the most important of which are called the Zn loop and the electrostatic loop. Each subunit also contains a zinc (Zn) ion and a copper (Cu) ion. The Zn loop serves as the binding site for the Zn ion, while the electrostatic loop stabilizes the binding of both ions. There is also an intramolecular disulfide bond that further stabilizes the structure by anchoring the Zn loop to the β-barrel.
In contrast, the most immature and unstable form of the enzyme, apoSOD12SH, exists as a monomer and does not contain any metal ions or disulfide bonds. This form of the enzyme is thought to be the toxic species that causes ALS (Rotunno and Bosco, 2013), so there is a clear need to learn more about its structure and other properties. Sekhar et al.—who are at the University of Toronto and the University of Waterloo—found that the Zn and electrostatic loops were much more flexible in apoSOD12SH than in Cu2Zn2SOD1S-S. However, the β-barrel structures of both forms are very similar.
If a molecule switches between its ground and excited states around 200–2000 times per second, and more than ∼0.5% of the sample is in the excited state at any one time, a form of NMR called CPMG relaxation dispersion provides detailed information about the excited states (Sekhar and Kay, 2013). A different NMR technique called CEST can be used when the molecules switches between conformations around 20–300 times per second (Fawzi et al., 2011; Vallurupalli et al., 2012). Sekhar et al. demonstrated that a combination of CPMG and CEST can elucidate multiple exchange processes between different conformations. Moreover, despite the underlying complexity of the processes, they were able to determine the thermodynamic, kinetic and structural properties of four short-lived excited states that are in equilibrium with the ground state of apoSOD12SH (Figure 1). Measurements of chemical shift differences indicated that two of the excited states were actually native conformations of the Cu2Zn2SOD1S-S dimer (Figure 1A).
Of particular interest are the conformational exchanges between apoSOD12SH and two non-native oligomers (Figure 1B). If factors such as mutations or modifications shift the equilibrium towards these two states, they might serve as starting points for the formation of more complex oligomers that could have a role in ALS.
By showing how NMR spin relaxation methods can reveal such details, the approach developed by Sekhar, Kay and co-workers has the potential to assist in the design of therapeutic molecules that target these oligomers.
Wild-type and mutant SOD1 share an aberrant conformation and a common pathogenic pathway in ALSNature Neuroscience 13:1396–1403.https://doi.org/10.1038/nn.2660
NMR studies of protein structure and dynamics - a look backwards and forwardsJournal of Magnetic Resonance 213:492–494.https://doi.org/10.1016/j.jmr.2011.08.010
NMR characterization of the dynamics of biomacromoleculesChemical Reviews 104:3623–3640.https://doi.org/10.1021/cr030413t
An emerging role for misfolded wild-type SOD1 in sporadic ALS pathogenesisFrontiers in Cellular Neuroscience 7:1–16.https://doi.org/10.3389/fncel.2013.00253
NMR paves the way for atomic level descriptions of sparsely populated, transiently formed biomolecular conformersProceedings of the National Academy of Sciences of USA 110:12867–12874.https://doi.org/10.1073/pnas.1305688110
Allosteric inhibition through suppression of transient conformational statesNature Chemical Biology 9:462–465.https://doi.org/10.1038/nchembio.1250
Studying “invisible” excited protein states in slow exchange with a major state conformationJournal of the American Chemical Society 134:8148–8161.https://doi.org/10.1021/ja3001419
Downloads (link to download the article as PDF)
Download citations (links to download the citations from this article in formats compatible with various reference manager tools)
Open citations (links to open the citations from this article in various online reference manager services)
Cellular aging is a multifactorial process that is characterized by a decline in homeostatic capacity, best described at the molecular level. Physicochemical properties such as pH and macromolecular crowding are essential to all molecular processes in cells and require maintenance. Whether a drift in physicochemical properties contributes to the overall decline of homeostasis in aging is not known. Here we show that the cytosol of yeast cells acidifies modestly in early aging and sharply after senescence. Using a macromolecular crowding sensor optimized for long-term FRET measurements, we show that crowding is rather stable and that the stability of crowding is a stronger predictor for lifespan than the absolute crowding levels. Additionally, in aged cells we observe drastic changes in organellar volume, leading to crowding on the µm scale, which we term organellar crowding. Our measurements provide an initial framework of physicochemical parameters of replicatively aged yeast cells.
PPP-family phosphatases such as PP1 have little intrinsic specificity. Cofactors can target PP1 to substrates or subcellular locations, but it remains unclear how they might confer sequence-specificity on PP1. The cytoskeletal regulator Phactr1 is a neuronally-enriched PP1 cofactor that is controlled by G-actin. Structural analysis showed that Phactr1 binding remodels PP1's hydrophobic groove, creating a new composite surface adjacent to the catalytic site. Using phosphoproteomics, we identified mouse fibroblast and neuronal Phactr1/PP1 substrates, which include cytoskeletal components and regulators. We determined high-resolution structures of Phactr1/PP1 bound to the dephosphorylated forms of its substrates IRSp53 and spectrin aII. Inversion of the phosphate in these holoenzyme-product complexes supports the proposed PPP-family catalytic mechanism. Substrate sequences C-terminal to the dephosphorylation site make intimate contacts with the composite Phactr1/PP1 surface, which are required for efficient dephosphorylation. Sequence specificity explains why Phactr1/PP1 exhibits orders-of-magnitude enhanced reactivity towards its substrates, compared to apo-PP1 or other PP1 holoenzymes.