Pamela J. E. Rowling

Hsp40 Interacts Directly with the Native State of the Yeast Prion Protein Ure2 and Inhibits Formation of Amyloid-like Fibrils

Ure2 is the protein determinant of the [URE3] prion phenotype in Saccharomyces cerevisiae and consists of a flexible N-terminal prion-determining domain and a globular C-terminal glutathione transferase-like domain. Overexpression of the type I Hsp40 member Ydj1 in yeast cells has been found to result in the loss of [URE3]. However, the mechanism of prion curing by Ydj1 remains unclear. Here we tested the effect of overexpression of Hsp40 members Ydj1, Sis1, and Apj1 and also Hsp70 co-chaperones Cpr7, Cns1, Sti1, and Fes1 in vivo and found that only Ydj1 showed a strong curing effect on [URE3]. We also investigated the interaction of Ydj1 with Ure2 in vitro. We found that Ydj1 was able to suppress formation of amyloid-like fibrils of Ure2 by delaying the process of fibril formation, as monitored by thioflavin T binding and atomic force microscopy imaging. Controls using bovine serum albumin, Sis1, or the human Hsp40 homologues Hdj1 or Hdj2 showed no significant inhibitory effect. Ydj1 was only effective when added during the lag phase of fibril formation, suggesting that it interacts with Ure2 at an early stage in fibril formation and delays the nucleation process. Using surface plasmon resonance and size exclusion chromatography, we demonstrated a direct interaction between Ydj1 and both wild type and N-terminally truncated Ure2. In contrast, Hdj2, which did not suppress fibril formation, did not show this interaction. The results suggest that Ydj1 inhibits Ure2 fibril formation by binding to the native state of Ure2, thus delaying the onset of oligomerization.

Shifting transition states in the unfolding of a large ankyrin repeat protein

The 33-amino-acid ankyrin motif comprises a β-turn followed by two anti-parallel α-helices and a loop and tandem arrays of the motif pack in a linear fashion to produce elongated structures characterized by short-range interactions. In this article we use site-directed mutagenesis to investigate the kinetic unfolding mechanism of D34, a 426-residue, 12-ankyrin repeat fragment of the protein ankyrinR. The data are consistent with a model in which the N-terminal half of the protein unfolds first by unraveling progressively from the start of the polypeptide chain to form an intermediate; in the next step, the C-terminal half of the protein unfolds via two pathways whose transition states have either the early or the late C-terminal ankyrin repeats folded. We conclude that the two halves of the protein unfold by different mechanisms because the N-terminal moiety folds and unfolds in the context of a folded C-terminal moiety, which therefore acts as a “seed” and confers a unique directionality on the process, whereas the C-terminal moiety folds and unfolds in the context of an unfolded N-terminal moiety and therefore behaves like a single-domain ankyrin repeat protein, having a high degree of symmetry and consequently more than one unfolding pathway accessible to it.

Toward Classification of BRCA1 Missense Variants Using a Biophysical Approach

Carriers of germ line mutations in breast cancer susceptibility gene BRCA1 have an increased risk of developing breast and ovarian cancers; missense mutations have, however, been difficult to assess for disease association. Here we have used a biophysical approach to classify these variants. We established an assay for measuring the thermodynamic stability of the BRCA1 BRCT domains and investigated the effects of 36 missense mutations. The mutations show a range of effects. Some do not change the stability, whereas others destabilize the protein by as much as 6 kcal mol−1; one-third of the mutants could not be expressed in soluble form in Escherichia coli, and we conclude that these destabilize the protein by an even greater amount. We tested several computer algorithms for their ability to predict the mutant effects and found that by grouping them into two classes (destabilizing by less than or more than 2.2 kcal mol−1), the algorithms could predict the stability changes. Importantly, with the exception of the few mutants located in the binding site, none showed a significant reduction in affinity for phosphorylated substrate. These results indicate that despite very large losses in stability, the integrity of the structure is not compromised by the mutations. Thus, the majority of mutations cause loss of function by reducing the proportion of BRCA1 molecules that are in the folded state and increasing the proportion of molecules that are unfolded. Consequently, small molecule stabilization of the structure could be a generally applicable preventative therapeutic strategy for rescuing many BRCA1 mutations.

Alcohol oxidase (AOX1) from Pichia pastoris is a novel inhibitor of prion propagation and a potential ATPase

Previous results suggest that methylotrophic yeasts may contain factors that modulate prion stability. Alcohol oxidase (AOX), a key enzyme in methanol metabolism, is an abundant protein that is specific to methylotrophic yeasts. We examined the effect of Pichia pastoris AOX1 on prion phenotypes in Saccharomyces cerevisiae. The S. cerevisiae prion states [PSI+] and [URE3] arise from aggregation of the proteins Sup35p and Ure2p respectively, and correlate with the ability of Sup35p and Ure2p to form amyloid‐like fibrils in vitro. We found that expression of P. pastoris AOX1 in S. cerevisiae had no effect on propagation of the [PSI+] prion, but inhibited propagation of [URE3]. Addition of AOX1 early in the time‐course of fibril formation inhibits Ure2p fibril formation in vitro. AOX1 has not previously been identified as an ATPase. However, we discovered that in addition to its flavin adenine dinucleotide‐dependent AOX activity, AOX1 possesses ATPase activity. This study identifies AOX1 as a novel prion inhibitory factor and a potential ATPase.

Protein stability versus function: effects of destabilizing missense mutations on BRCA1 DNA repair activity.

Mutations in breast cancer susceptibility gene BRCA1 (breast cancer early-onset 1) are associated with increased risk of developing breast and ovarian cancers. BRCA1 is a large protein of 1863 residues with two small structured domains at its termini: a RING domain at the N-terminus and a BRCT (BRCA1 C-terminus domain) repeat domain at the C-terminus. Previously, we quantified the effects of missense mutations on the thermodynamic stability of the BRCT domains, and we showed that many are so destabilizing that the folded functional state is drastically depopulated at physiological temperature. In the present study, we ask whether and how reduced thermodynamic stability of the isolated BRCT mutants translates into loss of function of the full-length protein in the cell. We assessed the effects of missense mutants on different stages of BRCA1-mediated DNA repair by homologous recombination using chicken lymphoblastoid DT40 cells as a model system. We found that all of the mutations, regardless of how profound their destabilizing effects, retained some DNA repair activity and thereby partially rescued the chicken BRCA1 knockout. By contrast, the mutation R1699L, which disrupts the binding of phosphorylated proteins (but which is not destabilizing), was completely inactive. It is likely that both protein context (location of the BRCT domains at the C-terminus of the large BRCA1 protein) and cellular environment (binding partners, molecular chaperones) buffer these destabilizing effects such that at least some mutant protein is able to adopt the folded functional state.

Subdomain architecture and stability of a giant repeat protein.

Tandem repeat proteins, which are widespread in the human genome, tend to exhibit high stability and favorable expression, and hence, they are emerging as promising protein scaffolds in alternative to antibodies in biotechnology. In order to investigate the origin of the stability of these proteins, we dissect the subdomain architecture of the giant repeat protein PR65/A, which comprises 15 α-helical HEAT repeats, using a series of truncations and deletions. We find that the N (HEAT 1-2) and the C (HEAT 14-15) subdomains are not capable of independent folding, but the addition of HEAT 13 to HEAT 14-15 results in an independently stable C-terminal subdomain (HEAT 13-15), which is in turn further stabilized by the inclusion of HEAT 12 (HEAT 12-15). We also further show that the stability of HEAT 13-15 is enhanced by its fusion to HEAT 1-2, and the artificial 5-HEAT-repeat protein thereby created (HEAT NC) behaves like a cooperative multidomain protein. We construct further variants, lacking one or both of the terminal subdomains, and find that such subdomains function as stabilizing caps within full-length PR65/A as in their absence, the central subdomain of the protein unfolds to form non-native β-sheet-like oligomers. Taken together, our results suggest that in full-length PR65/A, the more unstable regions within the central repeats are protected by the adjacent folded repeats, which thus act as gatekeepers by virtue of their greater stability.

Dissecting and reprogramming the folding and assembly of tandem-repeat proteins.

Studying protein folding and protein design in globular proteins presents significant challenges because of the two related features, topological complexity and co-operativity. In contrast, tandem-repeat proteins have regular and modular structures composed of linearly arrayed motifs. This means that the biophysics of even giant repeat proteins is highly amenable to dissection and to rational design. Here we discuss what has been learnt about the folding mechanisms of tandem-repeat proteins. The defining features that have emerged are: (i) accessibility of multiple distinct routes between denatured and native states, both at equilibrium and under kinetic conditions; (ii) different routes are favoured for folding compared with unfolding; (iii) unfolding energy barriers are broad, reflecting stepwise unravelling of an array repeat by repeat; (iv) highly co-operative unfolding at equilibrium and the potential for exceptionally high thermodynamic stabilities by introducing consensus residues; (v) under force, helical-repeat structures are very weak with non-co-operative unfolding leading to elasticity and buffering effects. This level of understanding should enable us to create repeat proteins with made-to-measure folding mechanisms, in which one can dial into the sequence the order of repeat folding, number of pathways taken, step size (co-operativity) and fine-structure of the kinetic energy barriers.

Diffuse transition state structure for the unfolding of a leucine-rich repeat protein.

Tandem-repeat proteins, such as leucine-rich repeats, comprise arrays of small structural motifs that pack in a linear fashion to produce elongated architectures. They lack contacts between residues that are distant in primary sequence, a feature that distinguishes them from the complex topologies of globular proteins. Here we have investigated the unfolding pathway of the leucine-rich repeat domain of the mRNA export protein TAP (TAPLRR) using Φ-value analysis. Whereas most of the tandem-repeat proteins studied to date have been found to unfold via a polarised mechanism in which only a small, localised number of repeats are structured in the transition state, the unfolding mechanism of TAPLRR is more diffuse in nature. In the transition state for unfolding of TAPLRR, three of the four LRRs are highly structured and non-native interactions are formed within the N-terminal α-helical cap and the first LRR. Thus, the α-helical cap plays an important role in which non-native interactions are required to provide a scaffold for the LRRs to pack against in the folding reaction.

Single-Molecule FRET Reveals Hidden Complexity in a Protein Energy Landscape

Summary Here, using single-molecule FRET, we reveal previously hidden conformations of the ankyrin-repeat domain of AnkyrinR, a giant adaptor molecule that anchors integral membrane proteins to the spectrin-actin cytoskeleton through simultaneous binding of multiple partner proteins. We show that the ankyrin repeats switch between high-FRET and low-FRET states, controlled by an unstructured “safety pin” or “staple” from the adjacent domain of AnkyrinR. Opening of the safety pin leads to unravelling of the ankyrin repeat stack, a process that will dramatically affect the relative orientations of AnkyrinR binding partners and, hence, the anchoring of the spectrin-actin cytoskeleton to the membrane. Ankyrin repeats are one of the most ubiquitous molecular recognition platforms in nature, and it is therefore important to understand how their structures are adapted for function. Our results point to a striking mechanism by which the order-disorder transition and, thereby, the activity of repeat proteins can be regulated.