Dynamics in Thermotoga neapolitana Adenylate Kinase: 15 N Relaxation and Hydrogen-Deuterium Exchange Studies of a Hyperthermophilic Enzyme Highly Active at 30 °C † Harini Krishnamurthy, ‡,§ Kim Munro, | Honggao Yan, ⊥ and Claire Vieille* ,⊥,# Program in Cell and Molecular Biology, Michigan State UniVersity, East Lansing, Michigan 48824, Queen’s Protein Function DiscoVery Facility, Queen’s UniVersity, Kingston, Ontario, Canada K7L-3N6, and Department of Biochemistry and Molecular Biology and Department of Microbiology and Molecular Genetics, Michigan State UniVersity, East Lansing, Michigan 48824 ReceiVed October 27, 2008; ReVised Manuscript ReceiVed January 16, 2009 ABSTRACT: Backbone conformational dynamics of Thermotoga neapolitana adenylate kinase in the free form (TNAK) and inhibitor-bound form (TNAK*Ap5A) were investigated at 30 °C using 15 N NMR relaxation measurements and NMR monitored hydrogen-deuterium exchange. With kinetic parameters identical to those of Escherichia coli AK (ECAK) at 30 °C, TNAK is a unique hyperthermophilic enzyme. These catalytic properties make TNAK an interesting and novel model to study the interplay between protein rigidity, stability, and activity. Comparison of fast time scale dynamics (picosecond to nanosecond) in the open and closed states of TNAK and ECAK at 30 °C reveals a uniformly higher rigidity across all domains of TNAK. Within this framework of a rigid TNAK structure, several residues located in the AMP-binding domain and in the core-lid hinge regions display high picosecond to nanosecond time scale flexibility. Together with the recent comparison of ECAK dynamics with those of hyperthermophilic Aquifex aeolicus AK (AAAK), our results provide strong evidence for the role of picosecond to nanosecond time scale fluctuations in both stability and activity. In the slow time scales, TNAK’s increased rigidity is not uniform but localized in the AMP-binding and lid domains. The core domain amides of ECAK and TNAK in the open and closed states show comparable protection against exchange. Significantly, the hinges framing the lid domain show similar exchange data in ECAK and TNAK open and closed forms. Our NMR relaxation and hydrogen-deuterium exchange studies therefore suggest that TNAK maintains high activity at 30 °C by localizing flexibility to the hinge regions that are key to facilitating conformational changes. Hyperthermophilic archaea and bacteria thrive at temper- atures above 80 °C, and their enzymes have unique structure-function properties of high stability and optimal activity at high temperatures. Hyperthermophilic proteins have been intensively studied for over 30 years to understand the molecular determinants and physical principles underly- ing protein thermostability and because thermostable en- zymes have potential biotechnological applications (see ref 1 for a comprehensive review). Many hyperthermophilic enzymes are optimally active above 80 °C, and they are mostly inactive at low tempera- tures (i.e., around 20-37 °C) (2-4). The catalytic rates of homologous mesophilic, thermophilic, and hyperthermophilic enzymes are usually comparable at the respective optimal growth temperatures of their source organisms (5). These properties of hyperthermophilic proteins have been explained by invoking the role of protein dynamics in catalysis. The reduced activity of hyperthermophilic enzymes at low temperatures appears to support the hypothesis that hyper- thermophilic proteins are more rigid than their mesophilic homologues at ambient temperatures. It is thought that enzyme motions required for activity become too slow and too restrained in hyperthermophilic enzymes at low temper- atures and that these proteins gain the flexibility required for optimal activity only at higher temperatures. Lack of activity at low temperatures need not be a consequence of stability at high temperatures, though, as shown by a number of laboratory-evolved thermostable enzymes that are catalyti- cally efficient at low temperatures (6). Also, as pointed out by Lazaridis et al. (7), an increase in flexibility should in fact contribute to the thermodynamic stability of the folded state through an increase in entropy. Understanding the role of protein dynamics in stability and activity requires ap- preciation of the fact that the protein energy landscape includes small fluctuations around the native state as well as transitions between conformational substates, including local and global unfolding events. These dynamic processes are spread across various time scales ranging from picosec- † H.K. was supported in part by a fellowship from the Michigan State University Center for Biological Modeling. * Corresponding author. Tel: (517) 884-5392. Fax: (517) 353-8957. E-mail: vieille@msu.edu. ‡ Program in Cell and Molecular Biology, Michigan State University. § Current address: Vollum Institute, Oregon Health and Science University, Portland, OR 97239. | Queen’s Protein Function Discovery Facility, Queen’s University. ⊥ Department of Biochemistry and Molecular Biology, Michigan State University. # Department of Microbiology and Molecular Genetics, Michigan State University. Biochemistry 2009, 48, 2723–2739 2723 10.1021/bi802001w CCC: $40.75  2009 American Chemical Society Published on Web 02/16/2009