Unfolding Transition State and Intermediates of the Tumor Suppressor p16 INK4a Investigated by Molecular Dynamics Simulations Gianluca Interlandi, Giovanni Settanni, and Amedeo Caflisch * Department of Biochemistry, University of Zu ¨ rich, Zu ¨ rich, Switzerland ABSTRACT The ankyrin repeat is one of the most common protein motifs and is involved in protein–protein interactions. It consists of 33 resi- dues that assume a -hairpin helix-loop-helix fold. Mutagenesis and kinetic experiments (-value anal- ysis of the folding transition state) have shown that the tumor suppressor p16 INK4a , a four-repeat pro- tein, unfolds sequentially starting from the two N-terminal repeats. Here, the flexibility of p16 INK4a at room temperature and its unfolding mechanism at high temperature have been investigated by mul- tiple molecular dynamics runs in explicit water for a total simulation time of 0.65 s. The transition state ensemble (TSE) of p16 INK4a was identified by monitoring both the deviation from the experimen- tal  values and sudden conformational changes along the unfolding trajectories. Conformations in the TSE have a mainly unstructured second repeat whereas the other repeats are almost completely folded. A rigid-body displacement of the first repeat involving both a rotation and translation is ob- served in all molecular dynamics simulations at high temperature. The Trp 15 , Pro 75 , and Ala 76 side- chains are more buried in the TSE than the native state. The sequential unfolding starting at the sec- ond repeat is in agreement with the mutagenesis studies whereas the displacement of the first repeat and the presence of nonnative interactions at the TSE are simulation results which supplement the experimental data. Furthermore, the unfolding tra- jectories reveal the presence of two on-pathway intermediates with partial -helical structure. Fi- nally, on the basis of the available experimental and simulation results we suggest that in modular pro- teins the shift of the folding TSE toward the native structure upon reduction of the number of tandem repeats is consistent with the Hammond effect. Proteins 2006;64:178 –192. © 2006 Wiley-Liss, Inc. Key words: ankyrin repeat proteins; flexibility; -value analysis; folding pathways; Hammond behavior; cancer INTRODUCTION Proteins consisting of a linear chain of homologous structural units, called repeats, are very common in na- ture and prevalently mediate protein–protein interac- tions. The ankyrin repeat consists of 33 amino acids forming a loop, a -turn, and two antiparallel -helices. 1 Several ankyrin repeats are clustered in a linear array to form an elongated structure that is stabilized predomi- nantly by short-range interactions between residues close in sequence. Moreover, the hydrophobic core has a toroidal shape unlike in globular proteins. p16 INK4a (Fig. 1), also referred to as multiple tumor suppressor 1 (Ref. 2) and abbreviated as p16 in this report, is a well-studied four-repeat protein, especially because of its direct link between cancer and cell cycle control. p16 regulates the proliferation of the cell by binding to cyclin D1 dependent kinases CDK4 and CDK6, and thus inhibit- ing them. The latter proteins are responsible for the inactivation of the retinoblastoma protein (Rb) through phosphorylation. If not phosphorylated, Rb blocks tran- scription by inhibiting a specific transcription factor such that the cell cannot proliferate. These functional relations are known as the p16/CDK4/CDK6/cycD1/Rbpathway, where p16 and Rb are two tumor suppressors. In fact, they are found mutated in cancerous tissues (for a review, see Lukas et al. 3 and Serrano 4 ). Experimentally, p16 was found to be marginally stable 2,5 and with a tendency to aggregate, which is drastically increased by single point mutations. 2,6 This might explain its deactivation in cancerous tissues, where it is often found mutated. Inactivation of p16 is second only to loss of p53 function as the most frequent event observed in human tumors. The nuclear magnetic resonance (NMR) solution structure first published in Byeon et al. 7 and later refined in Yuan et al. 8 revealed that the first helix of the second ankyrin repeat consists of only one turn. Further- more, compared with the helix bundles, the loops have a less-defined structure because of the conformational flex- ibility in these regions. Mutagenesis experiments have The Supplementary Material referred to in this article can be found at http://www.interscience.wiley.com/jpages/0887-3585/suppmat/ Grant sponsors: National Competence Center in Research (NCCR) in Structural Biology; Swiss National Science Foundation. Giovanni Settanni’s present address is MRC Center for Protein Engineering, Cambridge, UK. Correspondence to: Amedeo Caflisch, Department of Biochemis- try, University of Zu ¨ rich, 8057 Zu ¨ rich, Switzerland. E-mail: caflisch@bioc.unizh.ch Received 8 August 2005; Revised 9 November 2005; Accepted 8 December 2005 Published online 4 April 2006 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/prot.20953 PROTEINS: Structure, Function, and Bioinformatics 64:178 –192 (2006) © 2006 WILEY-LISS, INC.