Reduced Stability and Increased Dynamics in the Human Proliferating Cell Nuclear Antigen (PCNA) Relative to the Yeast Homolog

Proliferating Cell Nuclear Antigen (PCNA) is an essential factor for DNA replication and repair. PCNA forms a toroidal, ring shaped structure of 90 kDa by the symmetric association of three identical monomers. The ring encircles the DNA and acts as a platform where polymerases and other proteins dock to carry out different DNA metabolic processes. The amino acid sequence of human PCNA is 35% identical to the yeast homolog, and the two proteins have the same 3D crystal structure. In this report, we give evidence that the budding yeast (sc) and human (h) PCNAs have highly similar structures in solution but differ substantially in their stability and dynamics. hPCNA is less resistant to chemical and thermal denaturation and displays lower cooperativity of unfolding as compared to scPCNA. Solvent exchange rates measurements show that the slowest exchanging backbone amides are at the β-sheet, in the structure core, and not at the helices, which line the central channel. However, all the backbone amides of hPCNA exchange fast, becoming undetectable within hours, while the signals from the core amides of scPCNA persist for longer times. The high dynamics of the α-helices, which face the DNA in the PCNA-loaded form, is likely to have functional implications for the sliding of the PCNA ring on the DNA since a large hole with a flexible wall facilitates the establishment of protein-DNA interactions that are transient and easily broken. The increased dynamics of hPCNA relative to scPCNA may allow it to acquire multiple induced conformations upon binding to its substrates enlarging its binding diversity

Intrinsic Order and Disorder in the Bcl-2 Member Harakiri: Insights into Its Proapoptotic Activity

Harakiri is a BH3-only member of the Bcl-2 family that localizes in membranes and induces cell death by binding to prosurvival Bcl-xL and Bcl-2. The cytosolic domain of Harakiri is largely disorder with residual α-helical conformation according to previous structural studies. As these helical structures could play an important role in Harakiri's function, we have used NMR and circular dichroism to fully characterize them at the residue-atomic level. In addition, we report structural studies on a peptide fragment spanning Harakiri's C-terminal hydrophobic sequence, which potentially operates as a transmembrane domain. We initially checked by enzyme immunoassays and NMR that peptides encompassing different lengths of the cytosolic domain are functional as they bind Bcl-xL and Bcl-2. The structural data in water indicate that the α-helical conformation is restricted to a 25-residue segment comprising the BH3 domain. However, structure calculation was precluded because of insufficient NMR restraints. To bypass this problem we used alcohol-water mixture to increase structure population and confirmed by NMR that the conformation in both milieus is equivalent. The resulting three-dimensional structure closely resembles that of peptides encompassing the BH3 domain of BH3-only members in complex with their prosurvival partners, suggesting that preformed structural elements in the disordered protein are central to binding. In contrast, the transmembrane domain forms in micelles a monomeric α-helix with a population close to 100%. Its three-dimensional structure here reported reveals features that explain its function as membrane anchor. Altogether these results are used to propose a tentative structural model of how Harakiri works

Intrinsic Order and Disorder in the Bcl-2 Member Harakiri: Insights into Its Proapoptotic Activity

Harakiri is a BH3-only member of the Bcl-2 family that localizes in membranes and induces cell death by binding to prosurvival Bcl-xL and Bcl-2. The cytosolic domain of Harakiri is largely disorder with residual α-helical conformation according to previous structural studies. As these helical structures could play an important role in Harakiri's function, we have used NMR and circular dichroism to fully characterize them at the residue-atomic level. In addition, we report structural studies on a peptide fragment spanning Harakiri's C-terminal hydrophobic sequence, which potentially operates as a transmembrane domain. We initially checked by enzyme immunoassays and NMR that peptides encompassing different lengths of the cytosolic domain are functional as they bind Bcl-xL and Bcl-2. The structural data in water indicate that the α-helical conformation is restricted to a 25-residue segment comprising the BH3 domain. However, structure calculation was precluded because of insufficient NMR restraints. To bypass this problem we used alcohol-water mixture to increase structure population and confirmed by NMR that the conformation in both milieus is equivalent. The resulting three-dimensional structure closely resembles that of peptides encompassing the BH3 domain of BH3-only members in complex with their prosurvival partners, suggesting that preformed structural elements in the disordered protein are central to binding. In contrast, the transmembrane domain forms in micelles a monomeric α-helix with a population close to 100%. Its three-dimensional structure here reported reveals features that explain its function as membrane anchor. Altogether these results are used to propose a tentative structural model of how Harakiri works