The trans-to-cis proline isomerization in E. coli Trx folding is accelerated by trans prolines.

The conserved fold of thioredoxin (Trx)-like thiol/disulfide oxidoreductases contains an invariant cis-proline residue (P76 in Escherichia coli Trx) that is essential for Trx function and that is responsible for the folding rate-limiting step. E. coli Trx contains four additional prolines, which are all in the trans conformation in the native state. Notably, a recent study revealed that replacement of all four trans prolines in Trx by alanines (Trx variant Trx1P) further slowed the rate-limiting step 25-fold, indicating that one or several of the four trans prolines accelerate the trans-to-cis transition of P76 in Trx wild-type (wt). Here, we characterized the folding kinetics of Trx variants containing cisP76 and one or several of the natural trans prolines of Trx wt with NMR spectroscopy. First, we demonstrate that the isomerization reaction in Trx1P is a pure two-state transition between two distinct tertiary structures, in which all observed NMR resonances changes follow the same first-order kinetics. Moreover, we show that trans-P68 is the critical residue responsible for the faster folding of wt Trx relative to the single-proline (P76) variant Trx1P, as the two-proline variant Trx2P(P76P68) already folds seven times faster than Trx1P. trans-P34 also accelerates trans-to-cis isomerization of P76, albeit to a smaller extent. Overall, the results demonstrate that trans prolines can significantly modulate the kinetics of rate-limiting trans-to-cis proline isomerization in protein folding. Finally, we discuss possible mechanisms of acceleration and the potential significance of a protein-internal folding acceleration mechanism for Trx in a living cell.

Protein Allostery at Atomic Resolution.

Protein allostery is a phenomenon involving the long range coupling between two distal sites in a protein. In order to elucidate allostery at atomic resoluion on the ligand-binding WW domain of the enzyme Pin1, multistate structures were calculated from exact nuclear Overhauser effect (eNOE). In its free form, the protein undergoes a microsecond exchange between two states, one of which is predisposed to interact with its parent catalytic domain. In presence of the positive allosteric ligand, the equilibrium between the two states is shifted towards domain-domain interaction, suggesting a population shift model. In contrast, the allostery-suppressing ligand decouples the side-chain arrangement at the inter-domain interface thereby reducing the inter-domain interaction. As such, this mechanism is an example of dynamic allostery. The presented distinct modes of action highlight the power of the interplay between dynamics and function in the biological activity of proteins.

On the pH-dependence of α-synuclein amyloid polymorphism and the role of secondary nucleation in seed-based amyloid propagation.

The aggregation of the protein α-synuclein is closely associated with several neurodegenerative disorders and as such the structures of the amyloid fibril aggregates have high scientific and medical significance. However, there are dozens of unique atomic-resolution structures of these aggregates, and such a highly polymorphic nature of the α-synuclein fibrils hampers efforts in disease-relevant in vitro studies on α-synuclein amyloid aggregation. In order to better understand the factors that affect polymorph selection, we studied the structures of α-synuclein fibrils in vitro as a function of pH and buffer using cryo-EM helical reconstruction. We find that in the physiological range of pH 5.8-7.4, a pH-dependent selection between Type 1, 2, and 3 polymorphs occurs. Our results indicate that even in the presence of seeds, the polymorph selection during aggregation is highly dependent on the buffer conditions, attributed to the non-polymorph-specific nature of secondary nucleation. We also uncovered two new polymorphs that occur at pH 7.0 in phosphate-buffered saline. The first is a monofilament Type 1 fibril that highly resembles the structure of the juvenile-onset synucleinopathy polymorph found in patient-derived material. The second is a new Type 5 polymorph that resembles a polymorph that has been recently reported in a study that used diseased tissues to seed aggregation. Taken together, our results highlight the shallow amyloid energy hypersurface that can be altered by subtle changes in the environment, including the pH which is shown to play a major role in polymorph selection and in many cases appears to be the determining factor in seeded aggregation. The results also suggest the possibility of producing disease-relevant structure in vitro.

A structural rationale for reversible vs irreversible amyloid fibril formation from a single protein.

Reversible and irreversible amyloids are two diverging cases of protein (mis)folding associated with the cross-ß motif in the protein folding and aggregation energy landscape. Yet, the molecular origins responsible for the formation of reversible vs irreversible amyloids have remained unknown. Here we provide evidence at the atomic level of distinct folding motifs for irreversible and reversible amyloids derived from a single protein sequence: human lysozyme. We compare the 2.8 Å structure of irreversible amyloid fibrils determined by cryo-electron microscopy helical reconstructions with molecular insights gained by solid-state NMR spectroscopy on reversible amyloids. We observe a canonical cross-ß-sheet structure in irreversible amyloids, whereas in reversible amyloids, there is a less-ordered coexistence of ß-sheet and helical secondary structures that originate from a partially unfolded lysozyme, thus carrying a "memory" of the original folded protein precursor. We also report the structure of hen egg-white lysozyme irreversible amyloids at 3.2 Å resolution, revealing another canonical amyloid fold, and reaffirming that irreversible amyloids undergo a complete conversion of the native protein into the cross-ß structure. By combining atomic force microscopy, cryo-electron microscopy and solid-state NMR, we show that a full unfolding of the native protein precursor is a requirement for establishing irreversible amyloid fibrils.

An improved, time-efficient approach to extract accurate distance restraints for NMR2 structure calculation.

Exact nuclear Overhauser enhancement (eNOE) yields highly accurate, ensemble averaged 1H-1H distance restraints with an accuracy of up to 0.1 Šfor the multi-state structure determination of proteins as well as for nuclear magnetic resonance molecular replacement (NMR2) to determine the structure of the protein-ligand interaction site in a time-efficient manner. However, in the latter application, the acquired eNOEs lack the obtainable precision of 0.1 Šbecause of the asymmetrical nature of the filtered nuclear Overhauser enhancement spectroscopy (NOESY) experiment used in NMR2. This error is further propagated to the eNOE equations used to fit and extract the distance restraints. In this work, a new analysis method is proposed to obtain inter-molecular distance restraints from the filtered NOESY spectrum more accurately and intuitively by dividing the NOE cross peak by the corresponding diagonal peak of the ligand. The method termed diagonal-normalised eNOEs was tested on the data acquired by on the complex of PIN1 and a small, weak-binding phenylimidazole fragment. NMR2 calculations performed using the distances derived from diagonal-normalised eNOEs yielded the right orientation of the fragment in the binding pocket and produced a structure that more closely resembles the benchmark X-ray structure (2XP6) with an average heavy-atom root-mean-square deviation (RMSD) of 1.681 Šwith respect to it, when compared to the one produced with traditional NMR2 with an average heavy atom RMSD of 3.628 Å. This is attributed to the higher precision of the evaluated distance restraints.

Atomic resolution protein allostery from the multi-state structure of a PDZ domain.

Recent methodological advances in solution NMR allow the determination of multi-state protein structures and provide insights into structurally and dynamically correlated protein sites at atomic resolution. This is demonstrated in the present work for the well-studied PDZ2 domain of protein human tyrosine phosphatase 1E for which protein allostery had been predicted. Two-state protein structures were calculated for both the free form and in complex with the RA-GEF2 peptide using the exact nuclear Overhauser effect (eNOE) method. In the apo protein, an allosteric conformational selection step comprising almost 60% of the domain was detected with an "open" ligand welcoming state and a "closed" state that obstructs the binding site by changing the distance between the ß-sheet 2, α-helix 2, and sidechains of residues Lys38 and Lys72. The observed induced fit-type apo-holo structural rearrangements are in line with the previously published evolution-based analysis covering ~25% of the domain with only a partial overlap with the protein allostery of the open form. These presented structural studies highlight the presence of a dedicated highly optimized and complex dynamic interplay of the PDZ2 domain owed by the structure-dynamics landscape.