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Nuclear Magnetic Resonance Investigations of the 3-Dimensional Structure and Dynamics of Proteins in Solution

P.E. Wright, H.J. Dyson, A. Atkins, B. Duggan, M.P. Foster, M. Gearhart, S. Holmbeck, T.-H. Huang, B. Hudson, J. Laity, B. Lee, G. Legge, H.Y. Liu, J. Love, M. Martinez-Yamout, J. Pascual, G. Perez-Alvarado, J. Pikkemaat, I. Radhakrishnan, J. Xu, L. Zhu, T. Zor, M. Anderson-Landes, L.L. Tennant, J. Chung, D.A. Case, J. Gottesfeld, B. Cunningham, U. Hommel,* R. Evans,** M. Montminy***

* Novartis Pharmaceuticals, Basel, Switzerland
** The Salk Institute, La Jolla, CA
*** Harvard Medical School, Boston, MA

We use multidimensional nuclear magnetic resonance (NMR) spectroscopy to investigate the structures and dynamics of proteins in solution. Such studies are essential for understanding the mechanisms of action of these proteins and for elucidating structure-function relationships.

PROTEIN STRUCTURE DETERMINATION IN SOLUTION

Solution 3-dimensional structures have been determined for a number of proteins and protein complexes of molecular weight up to more than 20 kD. These include plastocyanin, thioredoxin, the human anaphylatoxin C3a, enzyme IIA^glc, myoglobin, rusticyanin, and the DNA-binding domains of several transcription factors, both free and bound to DNA. We are doing heteronuclear 3- and 4-dimensional NMR experiments with proteins labeled with ²H, ¹³C, or ¹⁵N to extend the NMR structure determination method to even larger proteins and protein complexes. In addition, new computational methods are being developed to facilitate structure determination and refinement.

TRANSCRIPTION FACTOR-DNA COMPLEXES

NMR methods are being used to determine the 3-dimensional structures and intramolecular dynamics of various DNA-binding motifs from eukaryotic transcriptional regulatory proteins, both free and complexed with the target DNA. Structures determined include proteins containing zinc finger motifs, the HMG box transcription factor lymphoid enhancer-binding factor 1, and the runt domain of the polyomavirus enhancer-binding protein 2.

Three-dimensional structures have been determined for the complex of zf1-3, a protein containing the N-terminal 3 of the 9 zinc fingers of transcription factor IIIA (TFIIIA), with the cognate 15-bp oligonucleotide duplex (Fig. 1). The 3 zinc fingers bind in the DNA major groove, thus validating parts of our earlier model of the TFIIIA-DNA complex deduced from biochemical experiments. The structures contain several novel features and show that prevailing models of DNA recognition, which assume that zinc fingers are independent modules that contact bases through a limited set of amino acids, are outmoded.

The repertoire of base contact residues is expanded in the zf1-3--DNA complex, and upon DNA binding, the protein forms an ordered globular structure with substantial protein-protein interactions between adjacent fingers. Dynamics measurements showed that the linker regions that connect the zinc finger domains are immobilized on binding to DNA. The linkers appear to play an active structural role in stabilization of the protein-DNA complex and together with finger-finger interactions contribute to high-affinity DNA binding. The NMR data provide evidence that many base contact residues remain conformationally flexible in the protein-DNA interface. Contributions to high-affinity binding come from both direct protein-DNA contacts and indirect protein-protein interactions associated with structural organization of the linkers and formation of packing interfaces between adjacent zinc fingers. We are now using residual dipolar couplings measured in dilute bicelle solutions to further refine the structure and thus better define the overall structure of the DNA in the complex.

In addition to its role in binding to and regulating the 5S RNA gene, TFIIIA also forms a complex with the 5S RNA transcript. The minimal set of zinc fingers required for high-affinity 5S RNA recognition consists of fingers 4--6, and the minimal region of the 5S RNA needed to bind these fingers has been mapped in J. Gottesfeld's laboratory, Department of Molecular Biology. NMR studies of the protein-RNA complex have commenced and should give important insights into the structural basis for 5S RNA recognition. In addition, zinc finger 6 from TFIIIA has been expressed, and its interactions with a small fragment of 5S RNA are being examined.

NMR studies of 2 alternative splice variants of the zinc finger protein of Wilms tumor are in progress. These variants differ only through insertion of 3 additional amino acids in the linker between fingers 3 and 4 yet have marked differences in their DNA-binding properties. Both variants have been expressed, and NMR studies of the structures and dynamics of the free proteins and of the protein-DNA complexes are under way.

In collaboration with R. Evans, the Salk Institute, the solution structure of the DNA-binding domain of the 9-cis retinoic acid receptor has been refined to high resolution. The results provide a basis for understanding the mechanisms of homodimerization and heterodimerization on different DNA recognition elements and of cooperativity in DNA binding. Binding of the receptor monomer to a single DNA half-site induces conformational changes that stabilize structure in the dimerization interface and promote dimer formation.

The DNA-binding domain of the human estrogen-related receptor has been expressed and labeled with ¹³C and ¹⁵N for NMR structural studies. This protein differs from the retinoic acid receptor and many other hormone receptors in that it binds DNA as a monomer. Determination of the structure and dynamics of the domain of the human estrogen-related receptor bound to DNA is in progress.

PROTEIN-PROTEIN INTERACTIONS IN TRANSCRIPTIONAL REGULATION

Activated transcriptional regulation in eukaryotes relies on protein-protein interactions between DNA-bound factors and coactivators that, in turn, interact with the basal transcription machinery. The cyclic AMP response element binding protein (CREB) activates transcription of target genes, in part through direct interactions with the KIX domain of the coactivator CREB-binding protein in a phosphorylation-dependent manner. In collaboration with M. Montminy, Harvard Medical School, we solved the structure of the complex formed by the phosphorylated kinase-inducible domain of CREB and the KIX domain of the CREB-binding protein.

This structure provides novel insights into the molecular basis of phosphoserine recognition and the coupling of folding and binding in the recognition of transcriptional activation domains. The phosphorylated kinase-inducible domain is largely unfolded in solution in the absence of the KIX domain but folds into a helical structure on binding to its target protein. Because many activation domains are intrinsically unstructured, similar folding transitions most likely play a general role in transcriptional activation. Ongoing work is directed toward mapping the interactions between KIX and other transcriptional activation domains and determining the 3-dimensional structures of other biologically important domains of the CREB-binding protein.

INTERACTIONS BETWEEN DOMAINS OF CELL ADHESION MOLECULES

We recently completed resonance assignments for the I (inserted) domain of lymphocyte function--associated antigen 1. The solution structure of the protein is now being determined by using a novel "molecular replacement" strategy that facilitates assignment of nuclear Overhauser effect cross peaks. Our interest in lymphocyte function--associated antigen 1 is in understanding its interactions with its physiologic partner, intracellular adhesion molecule 1. Domains of intracellular adhesion molecule 1 have been cloned and expressed in bacteria in preparation for structural studies. In parallel, we are collaborating with B. Cunningham, Department of Neurobiology, to probe the interactions between the immunoglobulin-like domains of the neural cell adhesion molecule. These projects illustrate the excellent sensitivity of the NMR method as a tool to probe protein-protein interactions: the NMR spectrum is used directly to map the sites of interaction, once assignments are available.

PUBLICATIONS

Chen, Y., Case, D.A., Reizer, J., Saier, M.H., Jr., Wright, P.E. High-resolution solution structure of Bacillus subtilis IIA^glc. Proteins 31:258, 1998.

Foster, M.P., Wuttke, D.S., Clemens, K.R., Jahnke, W., Radhakrishnan, I., Tennant, L., Reymond, M., Chung, J., Wright, P.E. Chemical shift as a probe of molecular interfaces: NMR studies of DNA binding by three amino-terminal zinc finger domains from transcription factor IIIA. J. Biomol. NMR, in press.

Foster, M.P., Wuttke, D.S., Radhakrishnan, I., Case, D.A., Gottesfeld, J.M., Wright, P.E. Domain packing and dynamics in the DNA complex of the amino terminal zinc fingers of transcription factor IIIA. Nature Struct. Biol. 4:605, 1997.

Gippert, G.P., Wright, P.E., Case, D.A. Distributed torsion angle grid search in high dimensions: A systematic approach to NMR structure determination. J. Biomol. NMR, in press.

Holmbeck, S.M.A., Foster, M.P., Casimiro, D.R., Sem, D., Dyson, H.J., Wright, P.E. High-resolution solution structure of the retinoid X receptor DNA-binding domain. J. Mol. Biol., in press.

Kriwacki, R.W., Wu, J., Tennant, L., Wright P.E., Siuzdak, G. Probing protein structure using biochemical and biophysical methods: Proteolysis, MALDI mass analysis, HPLC, and gel-filtration chromatography of p21 Wafl/Cipl/Sdi11. J. Chromatogr. 777:23, 1997.

Markley, J.L., Arata, Y., Bax, A., Hilbers, C.W., Kaptein, R., Sykes, B.D., Wright, P.E., Wüthrich, K. Recommendations for the presentation of NMR structures of proteins and nucleic acids. IUPAC-IUPAB Interunion Task Group. Pure Appl. Chem. 70:117, 1998.

Radhakrishnan, I., Perez-Alvarado, G., Parker, D., Dyson, H.J., Montminy, M., Wright, P.E. Solution structure of the KIX domain of CBP bound to the transactivation domain of CREB: A model for activator:coactivator interactions Cell 91:741, 1997.

Radhakrishnan, I., Perez-Alvarado, G.C., Dyson, H.J., Wright, P.E. Conformational preferences in the Ser133-phosphorylated and non-phosphorylated forms of the kinase inducible transactivation domain of CREB. FEBS Lett., in press.

Wuttke, D.S., Foster, M.P., Case, D.A., Gottesfeld, J.M., Wright, P.E. Solution structure of the first three zinc fingers of TFIIIA bound to the cognate DNA sequence: Determinants of affinity and sequence specificity. J. Mol. Biol. 273:183, 1997.

Zhu, L., Dyson, H.J., Wright, P.E. A NOESY-HSQC simulation program SPIRIT. J. Biomol. NMR 11:17, 1998.

Folding of Proteins and Protein Fragments

P.E. Wright, H.J. Dyson, Y. Bai, S. Cavagnero, D. Donne, D. Eliezer, C. Garcia-Gonzalez, V. Tsui, J. Viles, O. Zhang, M. Reymond, J. Chung, S. Lahrichi, L.L. Tennant

The molecular mechanism by which proteins fold into their 3-dimensional structures remains one of the most important unsolved problems in structural biology. Nuclear magnetic resonance (NMR) spectroscopy is uniquely suited to provide information on the structure of transient intermediates formed during protein folding. We used NMR methods to show that many peptide fragments of proteins tend to adopt folded conformations in water solution. The observation of transiently populated folded structures, including reverse turns, helices, nascent helices, and hydrophobic clusters, in water solutions of short peptides has important implications for initiation of protein folding. Formation of elements of secondary structure probably plays an important role in the initiation of protein folding by reducing the number of conformations that must be explored by the polypeptide chain and by directing subsequent folding pathways.

APOMYOGLOBIN FOLDING PATHWAY

A major program in our laboratory is to establish a structural and mechanistic description of the folding pathway of apomyoglobin. We used quenched-flow pulse-labeling methods in conjunction with 2-dimensional NMR spectroscopy to map the kinetic folding pathway of the wild-type protein. With these methods, we showed that an intermediate in which the A, G, and H helices adopt hydrogen-bonded secondary structure is formed within 6 msec of the initiation of refolding. Folding then proceeds by stabilization of structure in the B helix and then in the C and E helices. We are using carefully selected myoglobin mutants and both optical stopped-flow spectroscopy and NMR methods to further probe the kinetic folding pathway. For at least 2 of the mutants studied, the changes in amino acid sequence resulted in changes in the folding pathway of the protein.

An important issue in studies of myoglobin folding is the structure of unfolded and partly folded forms of the protein. Apomyoglobin provides a unique opportunity for detailed characterization of the structure and dynamics of a protein-folding intermediate. Conditions were determined under which the apomyoglobin molten globule intermediate is sufficiently stable for acquisition of multidimensional NMR spectra, and backbone resonance assignments based on ¹³C- and ¹⁵N-labeled protein are complete. Analysis of ¹³C and other chemical shifts and measurements of polypeptide dynamics are providing unprecedented insights into the structure of this state.

The A, G, and H helices and part of the B helix are folded and form the core of the molten globule. This core is stabilized by relatively nonspecific hydrophobic interactions that restrict the motions of the polypeptide chain. Fluctuating helical structure is formed in regions outside the core, although the amount of helix is low and the chain retains considerable flexibility. The F helix acts as a gate for heme-binding and only adopts stable structure in the fully folded holoprotein.

The acid-denatured state of apomyoglobin is an excellent model for the fluctuating local interactions that lead to the transient formation of unstable elements of secondary structure and local hydrophobic clusters during the earliest stages of folding. We succeeded in fully assigning the polypeptide backbone resonances in the acid-denatured unfolded state of apomyoglobin. The NMR data show formation of helical secondary structure in the denatured state in regions that form the A and H helices in the folded protein and also reveal nonnative structure in the D- and E-helix regions.

Because the A and H regions adopt stabilized helical structure in the earliest detectable folding intermediate, these results lend strong support to folding models in which spontaneous formation of local elements of secondary structure plays a role in initiating formation of the A-[B]-G-H molten globule folding intermediate. This role has been tested directly by introducing mutations into the H-helix region that reduce the propensity of the region for spontaneous formation of helical structure. Although these mutations do not affect the overall rate of folding of apomyoglobin, they do decrease the rate of folding of the H helix and the rate of formation of the A-[B]-G-H intermediate.

The view of protein folding that results from our work on apomyoglobin is one in which collapse of the polypeptide chain to form increasingly compact states leads to progressive accumulation of secondary structure and increasing restriction of fluctuations in the polypeptide backbone (Fig. 1). Chain flexibility is greatest at the earliest stages of folding, in which transient elements of secondary structure and local hydrophobic clusters are formed. As the folding protein becomes increasingly compact, backbone motions become more restricted, the hydrophobic core is formed and extended, and nascent elements of secondary structure are progressively stabilized. The ordered tertiary structure characteristic of the native protein, with well-packed side chains and relatively low-amplitude local dynamics, appears to form rather late in folding.

PLASTOCYANIN FOLDING

Structural characterization of an unfolded state of the ß-sheet protein apoplastocyanin is also in progress. Apoplastocyanin offers a unique opportunity to study an unfolded protein under nondenaturing conditions, because it forms an unfolded state at neutral pH under low-salt conditions. Extensive resonance assignments have been completed, revealing that the unfolded polypeptide has a marked propensity to populate the ß-region of dihedral angle space.

FRAGMENTS OF PRION PROTEINS

A number of proteins are unfolded or only partly folded before they bind to their ligand or substrate. Although scientists have known for many years that this fact applies to peptide hormones, we now realize that certain transcription factors, cyclin-dependent kinase inhibitors, and other proteins are unfolded in the absence of their natural receptors. In addition, folding has been implicated in a number of abnormalities, such as Alzheimer's disease and the prion diseases such as kuru and bovine spongiform encephalopathy (mad cow disease).

We are involved in elucidating the structure of some of the fragments of the prion protein. We showed that the dynamics of the N-terminal part of the prion molecule differ substantially from the dynamics of the C-terminal folded domain, indicating that the N-terminal part of the molecule is unfolded in solution. This promising new field has already indicated that the state of folding of the fragments will be crucial in the conversion between cellular and infectious forms of the prion protein.

PUBLICATIONS

Donne, D.G., Viles, J.H., Chung, J., Groth, D., Mehlhorn, I., Cohen, F.E., Prusiner, S.B., Wright, P.E., Dyson, H.J. Structure of the recombinant full-length Syrian hamster prion protein PrP(29-231): The N-terminus is highly flexible. Proc. Natl. Acad. Sci. U.S.A. 94:13452, 1997.

Dyson, H.J., Bolinger, L., Feher, V.A., Osterhout, J.J., Jr., Yao, J., Wright, P.E. Sequence requirements for stabilization of a peptide reverse turn in water solution: Proline is not essential for stability. Eur. J. Biochem., in press.

Dyson, H.J., Wright, P.E. Structural analysis of partly folded proteins. Nature Struct. Biol., in press.

Eliezer, D., Jennings, P.A., Dyson, H.J., Wright, P.E. Populating the equilibrium molten globule state of apomyoglobin under conditions suitable for structural characterization by NMR. FEBS Lett. 417:92, 1997.

Eliezer, D., Yao, J., Dyson, H.J., Wright, P.E. Structural and dynamic characterization of partially folded states of apomyoglobin and implications for protein folding. Nature Struct. Biol. 5:148, 1998.

Yao, J., Dyson, H.J., Wright, P.E. Chemical shift dispersion and secondary structure prediction in unfolded and partly-folded proteins. FEBS Lett. 419:285, 1997.