Cold Shock Domain of the Human Y-Box Protein YB-1. Backbone Dynamics and Equilibrium between the Native State and a Partially Unfolded State † Cathelijne P. A. M. Kloks, Marco Tessari, Geerten W. Vuister, and Cornelis W. Hilbers* NSRIM Centre, Faculty of Science, UniVersity of Nijmegen, ToernooiVeld 1, 6525 ED Nijmegen, The Netherlands ReceiVed March 10, 2004; ReVised Manuscript ReceiVed June 5, 2004 ABSTRACT: The three-dimensional structure of the central cold shock domain (CSD) of the human Y-box protein (YB-1 CSD) is virtually identical to those available for the bacterial cold shock proteins (Csp’s). We have further characterized YB-1 CSD by studying its dynamics by nuclear magnetic resonance. The observed structural similarity is reflected in the backbone dynamics, which for YB-1 CSD is very similar to that of the Escherichia coli protein CspA. The rotational correlation time of YB-1 CSD shows that it is a monomer. This indicates that the dimerization observed for the YB-1 protein is not caused by its CSD, but involves other parts of this protein. The YB-1 CSD is only marginally stable as are the mesophilic bacterial Csp’s. In contrast to the rapid two-state folding of the bacterial Csp’s, the formation of the native form of YB-1 CSD is slow and at least a three-state process. The NMR experiments revealed the presence of a second state of YB-1 CSD in equilibrium with the native form. The exchange rates from and to the folded state are in the order of 0.2 and 0.5 s -1 , respectively. Relaxation experiments indicated that the second state is a highly flexible, partly structured molecule. NMR relaxation measurements have gained a prominent position in studies of the internal mobility of biomacromol- ecules (1, 2). The high spectral resolution of the modern NMR spectrometers combined with multidimensional het- eronuclear techniques allows investigation of the relaxation behavior of individual atoms or groups of atoms in biom- acromolecules and thereby yields an unprecedented detailed picture of the dynamics of such molecules. The frequencies of the internal motions (including conformational changes) may vary considerably, i.e., from motions occurring in the second and millisecond up to the picosecond time scale. NMR relaxation experiments can be used to characterize these phenomena, although different NMR approaches are often required to determine the motions in the different frequency regions. Most frequently, 15 N-T 1 , 15 N-T 2 , and heteronuclear NOE ({ 1 H- 15 N}-NOE) experiments have been used to characterize the motion of the N-H N amide groups of the protein backbone. Current approaches used to interpret the results of such measurements are the so-called model-free approach (3, 4) and the (reduced) spectral density mapping method (5-9). The first method characterizes the internal motion in terms of an order parameter S 2 , which is a measure of the freedom of motion of a single atom or a group of atoms in a molecule. The second method provides the values of the spectral densities of the internal motions of an atom or a group of atoms at their respective Larmor frequencies. Exchange phenomena, as for example the exchange between different local conformational sites, are also reflected in the relaxation parameters. A particular form of conformational exchange, the transition between the folded and unfolded state of proteins, has so far received limited attention. The mixture of signals arising from the different forms complicate the spectrum interpretation and often result in increased spectral overlap, which makes relaxation studies inherently more difficult. A well-known example is the N-terminal SH3 domain of the protein drk (drkN SH3), which exists in equilibrium between a folded and unfolded state in aqueous solution (10, 11). Two other examples are the 131 residue- fragment of staphylococcal nuclease, ∆131∆ (12, 13), and the peripheral subunit-binding domain of pyruvate dehydro- genase multienzyme complex (14). In this article, we describe the backbone dynamics of the cold shock domain (CSD), which is the central, nucleic acid binding domain of the human Y-box protein, YB-1. Y-box proteins are involved in transcriptional and translational regulation. The general domain structure of this class of proteins is presented in Figure 1a, together with the consensus sequence of their CSDs. Previously, we derived the three- dimensional structure of the YB-1 CSD (15). The core of this domain consists of a five-stranded, antiparallel -barrel (Figure 1b,c) exhibiting a folding pattern characteristic of the oligonucleotide/oligosaccharide binding-fold (OB-fold) protein family (16). Just like drkN SH3, the folded form of YB-1 CSD coexists in a dynamic equilibrium with a second form, which we prove here to be largely unstructured. This form is referred to as the unfolded form indicated with U in the remainder of this article. We investigate the relaxation properties of these two forms and characterize their exchange properties. The results are compared with those of bacterial cold shock proteins (Csp’s) for which the three-dimensional † This research was supported by The Netherlands Foundation for Chemical Sciences with financial assistance from The Netherlands Organization for Scientific Research (NOW). * To whom correspondence should be addressed: Phone +31-24- 3652160. FAX: +31-24-3652112. E-mail: ceesh@sci.kun.nl. 10237 Biochemistry 2004, 43, 10237-10246 10.1021/bi049524s CCC: $27.50 © 2004 American Chemical Society Published on Web 07/14/2004