Protein Structure, Protein Folding, and Molecular Recognition

Our laboratory investigates interesting biological systems from structural, biophysical and biochemical perspectives. These studies further our understanding of the biology of the systems studied as well as provide insights into the fundamental nature of protein structure and function. The systems selected for study encompass several rapidly developing areas in biology and biochemistry, including telomere biology, viral replication, the estrogen receptor, and protein stability.

 

Structural Biology and Biochemistry of Telomeres

We are studying the structural biology and biochemical features of protein complexes important at telomeres. Telomeres are the specialized nucleoprotein complexes that cap eukaryotic chromosomes, protecting chromosome ends from unregulated degradation and end-to-end fusion. Telomeres also modulate the replicative potential of the cell by acting as substrates for the enzyme telomerase. The maintenance and replication of telomeres is currently under intense study because of the role telomeres play in tumorigenesis, the immortalization of human cell lines, and human aging. Cdc13 is a single-stranded telomeric DNA-binding protein identified genetically from S. cerevisiae. This essential protein protects the ends of telomeres from degradation and acts as both a positive and negative regulator of telomerase activity. Cdc13 fulfills both of these important roles through localization to the 3’ single-stranded telomeric end, and mediating protein/protein interactions with relevant complexes to the telomere. Consistent with this role, Cdc13 contains a single-stranded DNA (ssDNA) binding domain that we found exhibits an unusually high affinity - 3 pM - for ssDNA.

Recently, we solved the high-resolution structure of the DNA-binding domain of the essential yeast protein Cdc13 bound to its single-stranded DNA substrate, and have characterized the thermodynamic basis for high affinity and sequence-specific binding. The OB fold structure observed in Cdc13 links this protein to other functionally but not sequentially related proteins, including proteins implicated in telomere capping in several eukaryotes, including humans. The combination of structural and functional similarity between Cdc13 and telomere end-binding proteins from other distantly related eukaryotes suggests that mechanisms of telomeric end protection are widely conserved. The single-stranded DNA adopts a completely extended and irregular conformation in the complex with several of the aromatic groups stacking directly on a base in the DNA. We have used site-directed alanine mutagenesis to investigate the thermodynamic contribution of each sidechain identified at the interface to ssDNA binding and identified a "hotspot" of binding affinity corresponds precisely to the bases that are least tolerant to substitution and may provide a mechanism by which Cdc13 recognizes the heterogeneous sequences present at yeast telomeres. This series of alanine mutants will be used to better understand the in vivo role of Cdc13, and the effect of attenuating the affinity of Cdc13 on telomeres in yeast is currently being tested with our collaborator Vicki Lundblad.

Figure 1: Ribbon diagram of the stucture of the Cdc13-DBD.  Elements of secondary structure are labeled.

 

Viral Replication Proteins

We have solved the solution structure of the poliovirus 3A protein, a critical player in viral replication and immune suppression (Figure 2). Viruses from the Picornaviridae family are extremely successful and infect a wide spectrum of hosts, ranging from insects to humans. Members of the family include human rhinovirus (common cold), hepatitis A virus, poliovirus, the coxsackieviruses (chronic and acute heart disease), and echoviruses (neonatal lethal encephalitis) as well as important livestock viruses, including the causative agent of foot-and-mouth disease. Poliovirus is the most well-characterized member of this family, and serves as the paradigm for understanding this family of viruses. During infection, a membrane-bound RNA replication complex is formed that replicates the viral genome for further translation, replication, and packaging into viral prodigy. Poliovirus 3A protein is a critical component of this viral replication complex and is the target of an antiviral drug that blocks viral replication. 3A also inhibits host cell protein trafficking, a function which may play a key part in host immune evasion.

Figure 2:  Ribbon diagram of the structure of 3A.  Residues at the dimer interface are shown.

 

The 3A protein is a symmetric dimer with two helical hairpin monomers connected at a hydrophobic interface, and has no close structural relatives. This dimerization may be involved in the organization of the replication complex. Surprisingly, the N- and C- termini of 3A are unstructured, an interesting discovery as previous data implicates the N-terminus as being important in 3A functions. Mutations in this region abrogate the ability of the virus to replicate, implying that this region folds upon binding its as yet unidentified biological partner. The structure also reveals a patch of conserved negative charge that may be important for 3A function.

 

Therapeutic and Environmental Ligand Binding to the Estrogen Receptor

We are developing a program to investigate agonist and antagonist binding to the ligand-binding domain (LBD) of the estrogen receptor (ER) by taking advantage of recent advances in NMR spectroscopy that have made it possible to study systems of this size (56 kDa). The ER is an essential nuclear hormone receptor, distributed in breast, bone, and liver tissue, the organs of the reproductive system, and the cardiovascular system. Estrogens bind to activate the ER, which leads to the stimulation of transcription at specific genes. Through this receptor, estrogen regulates the development of reproductive tissues in both males and females and stimulates skeletal maturation. Intriguingly, the ER binds several types of compounds, including compounds that are quite distinct from its natural ligand. Antiestrogens and partial antiestrogens bind tightly to the ER but fail to activate transcription; these compounds, e.g., tamoxifen, are in widespread use for the treatment of breast cancer. In addition, a variety of xenobiotic compounds have been found to act as estrogen mimics and alter reproductive function and development. We are interested in the relationship between exposure to xenobiotic compounds and increased breast cancer risk.

 

We are using chemical shift mapping to correlate the changes that occur upon ligand binding with structural changes observed in the crystal structures of ER-LBD complexes with ligands. We have expressed, purified to high homogeneity, and uniformly isotopically labeled the ER-LBD with 2H and 15N. Comparison of the high-resolution spectra of ligand-free, estradiol-bound and tamoxifen-bound states of the receptor reveal several intriguing differences between these states of the protein. The availability of an 800 MHz NMR spectrometer in Boulder will allow us to obtain assignment data needed to pinpoint the sites of change onto the structure.

 

Protein Stability using Fragment Complementation

We are using the fragment complementation of proteins to analyze the interactions that stabilize protein structures. The SH2 domain retains the abililty to fold and function as a non-covalent complex when cleaved within the peptide backbone. Using this reassembly as an assay for folding, we have examined the sequence elements necessary for stable protein folding. Our NMR studies revealed that the individual fragments are completely unstructured, while the non-covalent fragment complex exhibits a native-like NMR spectrum. By replacing each amino acid in the smaller fragments with alanine and screening for complementation activity, we were able to determine the contribution of each site to overall stability. The effects on binding affinity of the replacement of a side chain with alanine span a wide dynamic range, varying from complete loss of binding to slightly enhanced binding affinity relative to wild type (Figure 3). The alanine substitutions that abolished folding correlate well with highly conserved residues that are either integrally involved in core packing or are found at the interface between fragments. Surprisingly, however, a non-conserved surface-exposed aspartic acid had a significant effect on complementation. This aspartate residue appears to be necessary to satisfy electrostatic interactions at the surface of the protein, providing evidence that both favorable and unfavorable surface charge interactions participate in determining protein stability.

 

 

Figure 3:  The effect of alanine scanning on increasing folding ability mapped on the SH2 backbone.

Selected References:

D. L. Theobald, R. M. Mitton-Fry and D. S. Wuttke, "Nucleic Acid Recognition by OB-Fold Proteins," Annu. Rev. Biophys. and Biochem., invited review, submitted.

 

 E. M. Anderson and D. S. Wuttke, "Site-Directed Mutagenesis Reveals the Thermodynamic Requirements for Single-Stranded DNA Recognition by The Telomere-Binding Protein Cdc13," submitted.

 

 L. W. Glustrom and D. S. Wuttke, "Dichlorodiphenyltrichloroethane (DDT), Dichlorodiphenyldichloroethylene (DDE) and Breast Cancer: The Potential Role of Hydroxylated Metabolites," submitted.

 

 L. W. Glustrom, R. M. Mitton-Fry and D. S. Wuttke, "Environmental Estrogens and Breast Cancer: The Importance of Hydroxylated Intermediates," reviewed letter, J. Natl. Cancer Institute, 2002, 94, 68-69.

 

D. D. Ojennus, S. E. Lehto and D. S. Wuttke, “Delineation of Essential Sequence Elements for the Reconstitution of an SH2 Domain from Constituent Peptide Fragments,” submitted.

E. M. Anderson, W. A. Halsey and D. S. Wuttke, “Delineation of the High-Affinity Single-Stranded Telomeric DNA-Binding Domain of S. cerevisiae Cdc13,” Nucleic Acids Research, in press.

R. M. Mitton-Fry and D. S. Wuttke, “¹H, ¹³C and ¹⁵N resonance assignments of the DNA-binding domain of the essential protein Cdc13 complexed with single-stranded telomeric DNA,” J. Biomol. NMR, 2002, 22, 379 – 380

R. M. Mitton-Fry, E. M. Anderson, T. R. Hughes, V. Lundlblad, D. S. Wuttke, “Conserved Structure for Single-Stranded Telomeric DNA Recognition,” Science, 2002, 296, 145 - 147. . [Abstract][Full Text]

D. D. Ojennus, M. R. Fleissner and D. S. Wuttke, “Reconstitution of a Native-Like SH2 Domain from Disordered Peptide Fragments Examined by Multidimensional Heteronuclear NMR,” Protein Science, 2001, 10, 2162 - 2175.

D. D. Ojennus, R. M. Mitton-Fry, and D. S. Wuttke, “Induced Alignment and Measurement of Dipolar Couplings in an SH2 Domain Through Direct Binding with Filamentous Phage,” J. Biomol. NMR, 1999, 14, 175-179.