p27(Kip1) contributes to cell-cycle regulation by inhibiting cyclin-dependent kinase (Cdk) activity. The p27 Cdk-inhibition domain has an ordered conformation comprising an alpha-helix, a 3(10) helix, and beta-structure when bound to cyclin A-Cdk2. In contrast, the unbound p27 Cdk-inhibition domain is intrinsically disordered (natively unfolded) as shown by circular dichroism spectroscopy, lack of chemical-shift dispersion, and negative heteronuclear nuclear Overhauser effects. The intrinsic disorder is not due to the excision of the Cdk-inhibition domain from p27, since circular dichroism spectra of the full-length protein are also indicative of a largely unfolded protein. Both the inhibition domain and full-length p27 are active as cyclin A-Cdk2 inhibitors. Using circular dichroism and proline mutagenesis, we demonstrate that the unbound p27 Cdk-inhibition domain is not completely unfolded. The domain contains marginally stable helical structure that presages the alpha-helix, but not the 3(10) helix, adopted upon binding cyclin A-Cdk2. Increasing or reducing the stability of the partially preformed alpha-helix in the isolated p27 domain with alanine or proline substitutions did not affect formation of the p27-inhibited cyclin A-Cdk2 complex in energetic terms. However, stabilization of the helix with alanine hindered kinetically the formation of the inhibited complex, suggesting that p27 derives a kinetic advantage from intrinsic structural disorder.

Since the advent of genome-wide small interfering RNA screening, large numbers of cellular cofactors important for viral infection have been discovered at a rapid pace, but the viral targets and the mechanism of action for many of these cofactors remain undefined. One such cofactor is cyclophilin A (CyPA), upon which hepatitis C virus (HCV) replication critically depends. Here we report a new genetic selection scheme that identified a major viral determinant of HCV's dependence on CyPA and susceptibility to cyclosporine A. We selected mutant viruses that were able to infect CyPA-knockdown cells which were refractory to infection by wild-type HCV produced in cell culture. Five independent selections revealed related mutations in a single dipeptide motif (D316 and Y317) located in a proline-rich region of NS5A domain II, which has been implicated in CyPA binding. Engineering the mutations into wild-type HCV fully recapitulated the CyPA-independent and CsA-resistant phenotype and four putative proline substrates of CyPA were mapped to the vicinity of the DY motif. Circular dichroism analysis of wild-type and mutant NS5A peptides indicated that the D316E/Y317N mutations (DEYN) induced a conformational change at a major CyPA-binding site. Furthermore, nuclear magnetic resonance experiments suggested that NS5A with DEYN mutations adopts a more extended, functional conformation in the putative CyPA substrate site in domain II. Finally, the importance of this major CsA-sensitivity determinant was confirmed in additional genotypes (GT) other than GT 2a. This study describes a new genetic approach to identifying viral targets of cellular cofactors and identifies a major regulator of HCV's susceptibility to CsA and its derivatives that are currently in clinical trials.

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The 1H, 13C, 15N and 31P random-coil chemical shifts and phosphate pKa values of phosphorylated amino acids pSer, pThr and pTyr in the protected peptide Ac-Gly-Gly-X-Gly-Gly-NH2 have been obtained in water at 25 degrees C over the pH range 2 to 9. Analysis of ROESY spectra indicates that the peptides are unstructured. Phosphorylation induces changes in random-coil chemical shifts, some of which are comparable to those caused by secondary structure formation, and are therefore significant in structural analyses based on the chemical shift.

Amyloid-β (Aβ) peptide aggregation is known to play a central role in the etiology of Alzheimer’s disease (AD). Among various aggregates, low-molecular weight soluble oligomers of Aβ are increasingly believed to be the primary neurotoxic agents responsible for memory impairment. Anionic interfaces are known to influence the Aβ aggregation process significantly. Here, we report the effects of interfaces formed by medium-chain (C9–C12), saturated non-esterified fatty acids (NEFAs) on Aβ42 aggregation. NEFAs uniquely affected Aβ42 aggregation rates that depended on both the ratio of Aβ:NEFA as well the critical micelle concentration (CMC) of the NEFAs. More importantly, irrespective of the kind of NEFA used, we observed that two distinct oligomers, 12–18 mers and 4–5 mers were formed via different pathway of aggregation under specific experimental conditions: (i) 12–18 mers were generated near the CMC in which NEFAs augment the rate of Aβ42 aggregation towards fibril formation, and, (ii) 4–5 mers were formed above the CMC, where NEFAs inhibit fibril formation. The data indicated that both 12–18 mers and 4–5 mers are formed along an alternate pathway called ‘off-pathway’ that did not result in fibril formation and yet have subtle structural and morphological differences that distinguish their bulk molecular behavior. These observations, (i) reflect the possible mechanism of Aβ aggregation in physiological lipid-rich environments, and (ii) reiterate the fact that all oligomeric forms of Aβ need not be obligatory intermediates of the fibril formation pathway.