DOI: 10.1126/science.1157092 1471 (2008) 320, Science et al. Oliver F. Lange, in Solution Revealed from an RDC-Derived Ubiquitin Ensemble Recognition Dynamics Up to Microseconds This copy is for your personal, non-commercial use only. clicking here. colleagues, clients, or customers by you can order high-quality copies for your If you wish to distribute this article to others, here. following the guidelines can be obtained by Permission to republish or repurpose articles or portions of articles (this information is current as of February 17, 2010 ): The following resources related to this article are available online at www.sciencemag.org http://www.sciencemag.org/cgi/content/full/320/5882/1471 version of this article at: including high-resolution figures, can be found in the online Updated information and services, http://www.sciencemag.org/cgi/content/full/320/5882/1471/DC1 can be found at: Supporting Online Material found at: can be related to this article A list of selected additional articles on the Science Web sites http://www.sciencemag.org/cgi/content/full/320/5882/1471#related-content http://www.sciencemag.org/cgi/content/full/320/5882/1471#otherarticles 11 of which can be accessed for free: cites 44 articles, This article 100 article(s) on the ISI Web of Science. cited by This article has been http://www.sciencemag.org/cgi/content/full/320/5882/1471#otherarticles 10 articles hosted by HighWire Press see: cited by This article has been http://www.sciencemag.org/cgi/collection/biochem Biochemistry subject collections: This article appears in the following registered trademark of AAAS. is a Science 2008 by the American Association for the Advancement of Science all rights reserved. The title Copyright American Association for the Advancement of Science, 1200 New York Avenue NW, Washington, DC 20005. (print ISSN 0036-8075 online ISSN 1095-9203) is published weekly, except the last week in December, by the Science on February 17, 2010 www.sciencemag.org Downloaded from

RecognitionDynamicsUptoMicroseconds Revealed from an RDC-Derived Ubiquitin Ensemble in Solution Oliver F. Lange,1* Nils-Alexander Lakomek,2* Christophe Far��s,2 Gunnar F. Schr��der,1 Korvin F. A. Walter,2 Stefan Becker,2 Jens Meiler,3 Helmut Grubm��ller,1 Christian Griesinger,2��� Bert L. de Groot1��� Protein dynamics are essential for protein function, and yet it has been challenging to access the underlying atomic motions in solution on nanosecond-to-microsecond time scales. We present a structural ensemble of ubiquitin, refined against residual dipolar couplings (RDCs), comprising solution dynamics up to microseconds. The ensemble covers the complete structural heterogeneity observed in 46 ubiquitin crystal structures, most of which are complexes with other proteins. Conformational selection, rather than induced-fit motion, thus suffices to explain the molecular recognition dynamics of ubiquitin. Marked correlations are seen between the flexibility of the ensemble and contacts formed in ubiquitin complexes. A large part of the solution dynamics is concentrated in one concerted mode, which accounts for most of ubiquitin���s molecular recognition heterogeneity and ensures a low entropic complex formation cost. Pfrom rotein function relies on structural pro- tein dynamics, with time scales ranging picoseconds to beyond seconds. For molecular recognition, for example, proteins adapt their structure to different binding partners, often exhibiting large structural heterogeneity. In the past 30 years, atomic information on many dynamical processes has been accumulated from a broad variety of techniques (1, 2). Nuclear magnetic resonance (NMR) relaxation has been used to quantitatively probe protein dynamics at the fast end (picoseconds to nanoseconds) as well as in a much slower range (microseconds to mil- liseconds) of this broad spectrum of time scales (3���6). Relaxation of nuclear magnetization is caused by fluctuations of magnetic interactions between nuclei resulting from the nanosecond rotational tumbling of the molecule and internal dynamics. The amplitudes of these motions are expressed as so-called Lipari-Szabo order pa- rameters S LS 2 (7). Internal dynamics slower than the rotational tumbling time tc have no impact on the overall fluctuation of the magnetic in- teractions. Therefore, S LS 2 order parameters reflect only sub-tc motions, at the fast end of time scales. The slow range of time scales is accessible by relaxation dispersion measurements, based on the stochastic fluctuations of isotropic chem- ical shifts, which are independent of rotational tumbling (3, 5). Conformational heterogeneity slower than 10 ms can be directly observed as peak splitting in NMR spectra. For backbone amides, motions faster than 50 ms do not result in sufficient line broadening to be detectable for relaxation dispersion measurements. These mea- surements therefore probe motions slower than about 50 ms up to about 10 ms and have been used to characterize major structural changes and enzymatic reactions (6, 8). Except for cer- tain favorable cases (9), it is, however, difficult to translate these fluctuations into ensembles of structures. Therefore, relaxation-based ensem- bles of solution structures take only motions faster than tc into account: They are limited to sub-tc dynamics (10, 11). These sub-tc mo- tions are typically much smaller than the struc- tural changes involved in molecular recognition and are likely to contribute mainly to the en- tropy of proteins (12���14). As a consequence, the structural heterogeneity observed in pro- tein complexes has frequently been assumed to be inaccessible to equilibrium fluctuations in solution, thus favoring induced-fit models (15, 16). RDCs probe supra-tc dynamics. RDCs are sensitive to motion from picoseconds to milli- seconds, which includes the previously in- visible time window between tc and 50 ms, which we will call supra-tc. Indeed, RDCs recorded for ubiquitin, as well as for the B1 domain of protein G, hint at substantial dy- namics between nanoseconds and microsec- onds (17���25). Here, we present a structural ensemble of ubiquitin based on an extensive 1Department of Theoretical and Computational Biophysics, Max Planck Institute for Biophysical Chemistry, Am Fassberg 11, 37077 G��ttingen, Germany. 2Department of NMR Based Structural Biology, Max Planck Institute for Biophysical Chem- istry, Am Fassberg 11, 37077 G��ttingen, Germany. 3 Vanderbilt University Center for Structural Biology, Nashville, TN 37212, USA. *These authors contributed equally to this work. ���To whom correspondence should be addressed. E-mail: cigr@nmr.mpibpc.mpg.de (C.G.) bgroot@gwdg.de (B.L.dG.) loop ��3�����2 10 20 30 40 0 0.5 1 1.5 2 2.5 X-ray structure C �� -RMSD [��] 0.7 0.8 0.9 1 Ssupra 2 K63 ��1�����3 loop loop ��1�����2 1 10 20 30 40 50 60 70 0 0.05 0.1 0.15 0.2 0.25 Residue RMSF (nm) A B C Fig. 1. Structure ensemble of ubiquitin. (A) Backbone trace of 40 randomly chosen structures from the EROS ensemble. Residues are colored by the amount of additional (supra-tc) mobility as compared with the Lipari-Szabo order parameters (Fig. 3C) Ssupra 2 = SEROS/SLS. 2 2 (B) For each x-ray structure (for numbering on the x axis, see table S3), the backbone RMSDs of residues 1 to 70 are shown for superpositions with each EROS structure (red dots) and each x-ray structure (black dots). The minimal RMSD for EROS structures (red line) and the maximal RMSD for x-ray structures (black line) are highlighted to guide the eye. (C) Ca root mean square fluctuations (RMSF) of EROS structures (red line) and of 46 known ubiquitin x-ray structures (black line). www.sciencemag.org SCIENCE VOL 320 13 JUNE 2008 1471 RESEARCH ARTICLES on February 17, 2010 www.sciencemag.org Downloaded from