Jack Liao

Single-stranded DNA mimicry in the p53 transactivation domain interaction with replication protein A

One of many protein–protein interactions modulated upon DNA damage is that of the single-stranded DNA-binding protein, replication protein A (RPA), with the p53 tumor suppressor. Here we report the crystal structure of RPA residues 1–120 (RPA70N) bound to the N-terminal transactivation domain of p53 (residues 37–57; p53N) and, by using NMR spectroscopy, characterize two mechanisms by which the RPA/p53 interaction can be modulated. RPA70N forms an oligonucleotide/oligosaccharide-binding fold, similar to that previously observed for the ssDNA-binding domains of RPA. In contrast, the N-terminal p53 transactivation domain is largely disordered in solution, but residues 37–57 fold into two amphipathic helices, H1 and H2, upon binding with RPA70N. The H2 helix of p53 structurally mimics the binding of ssDNA to the oligonucleotide/oligosaccharide-binding fold. NMR experiments confirmed that both ssDNA and an acidic peptide mimicking a phosphorylated form of RPA32N can independently compete the acidic p53N out of the binding site. Taken together, our data suggest a mechanism for DNA damage signaling that can explain a threshold response to DNA damage.

An NMR approach to structural proteomics

The influx of genomic sequence information has led to the concept of structural proteomics, the determination of protein structures on a genome-wide scale. Here we describe an approach to structural proteomics of small proteins using NMR spectroscopy. Over 500 small proteins from several organisms were cloned, expressed, purified, and evaluated by NMR. Although there was variability among proteomes, overall 20% of these proteins were found to be readily amenable to NMR structure determination. NMR sample preparation was centralized in one facility, and a distributive approach was used for NMR data collection and analysis. Twelve structures are reported here as part of this approach, which allowed us to infer putative functions for several conserved hypothetical proteins.

Interferon-Inducible Protein 16: Insight into the Interaction with Tumor Suppressor p53

IFI16 is a member of the interferon-inducible HIN-200 family of nuclear proteins. It has been implicated in transcriptional regulation by modulating protein-protein interactions with p53 tumor suppressor protein and other transcription factors. However, the mechanisms of interaction remain unknown. Here, we report the crystal structures of both HIN-A and HIN-B domains of IFI16 determined at 2.0 and 2.35 Å resolution, respectively. Each HIN domain comprises a pair of tightly packed OB-fold subdomains that appear to act as a single unit. We show that both HIN domains of IFI16 are capable of enhancing p53-DNA complex formation and transcriptional activation via distinctive means. HIN-A domain binds to the basic C terminus of p53, whereas the HIN-B domain binds to the core DNA-binding region of p53. Both interactions are compatible with the DNA-bound state of p53 and together contribute to the effect of full-length IFI16 on p53-DNA complex formation and transcriptional activation.

Preferential binding of IFI16 protein to cruciform structure and superhelical DNA.

Interferon (IFN)-inducible HIN-200 proteins play an important role in transcriptional regulation linked to cell cycle control, inflammation, autoimmunity and differentiation. IFI16 has been identified as a target of IFNα and γ and is a member of the HIN-200 protein family. Expression level of IFI16 is often decreased in breast cancers, implicating its role as a tumor suppressor. As a potent transcription factor, IFI16 possesses a transcriptional regulatory region, a PYD/DAPIN/PAAD region which associates with IFN response, DNA-binding domains and binding regions for tumor suppressor proteins BRCA1 and p53. It is also reported that IFI16 protein is capable of binding p53 and cMYC gene promoters. Here, we demonstrate that IFI16 protein binds strongly to negatively superhelical plasmid DNA at a native superhelix density, as evidenced by electrophoretic retardation of supercoiled (sc) DNA in agarose gels. Binding of IFI16 to supercoiled DNA results in the appearance of one or more retarded DNA bands on the gels. After removal of IFI16, the original mobility of the scDNA is recovered. By contrast, IFI16 protein binds very weakly to the same DNA in linear state. Using short oligonucleotide targets, we also detect a strong preference for IFI16 binding to cruciform DNA structure compared to linear DNA topology. Hence, this novel DNA-binding property of IFI16 protein to scDNA and cruciform structures may play critical roles in its tumor suppressor function.

The Central Region of BRCA1 Binds Preferentially to Supercoiled DNA

Abstract BRCA1 is a multifunctional tumor suppressor protein with implications in regulating processes such as cell cycle, transcription, DNA repair, and chromatin remodeling. The function of BRCA1 likely involves interactions with a vast number of proteins and likewise DNA. To this date there is only fragmentary evidence about BRCA1 binding to DNA. In this study, we provide detailed analyses of various BRCA1 protein constructs binding to linear and super-coiled (sc) DNAs. We demonstrate that the central region of human BRCA1 binds strongly to negatively sc plasmid DNA at a native superhelix density, as evidenced by electrophoretic retardation of sc DNA in agarose gels. At relatively low BRCA1:DNA ratios, binding of BRCA1 to sc DNA results in the appearance of one or more retarded DNA bands on the gels. After removal of BRCA1, the original mobility of the sc DNA is recovered. BRCA1 proteins at higher concentrations also bind to the same DNA but in linear state, leading to formation of a smeared retarded band. Our experiments not only demonstrate a preference for BRCA1 binding to sc DNA, but also show that the central region may contain at least two efficient DNA binding domains with strong affinity for sc DNA. The biological implications of the novel DNA binding activities of BRCA1 are discussed.

An assessment of the chiral environment created by adjacent D- and L-alanyl residues on a glycine unit within the tripeptide N-Ac-Ala-Gly-Ala-NHMe: An ab initio exploratory study

Ab initio Molecular Orbital computations were carried out on the tripeptide models, N-Ac-D-Ala-Gly-L-Ala-NHMe and NAc-L-Ala-Gly-L-Ala-NHMe at the RHF/3-21G level of theory. The topologies of conformational Potential Energy Surfaces were explored and analyzed. In addition, global and local minima on the Ramachandran Potential Energy Surfaces, E ¼ f ðf1;c1) and E ¼ f ðf3;c3) were identified and their geometries optimized. The nearest neighboring effects of D and L amino acids on the conformations of the central glycine residue were also compared. Seven stable minima were found for each system and two conformers (a and 1) were oppositely different due to the influence of the N-terminal D -o rL-alanyl residue. q 2003 Elsevier Science B.V. All rights reserved.

NMR structure of the hypothetical protein encoded by the YjbJ gene from Escherichia coli.

Here we describe the solution structure of YjbJ (gil418541) as part of a structural proteomics project on the feasibility of the high-throughput generation of samples from Escherichia coli for structural studies. YjbJ is a hypothetical protein from Escherichia coli protein of unknown function. It is conserved, showing significant sequence identity to four predicted prokaryotic proteins, also of unknown function (Figure 1A). These include gil16762921 from Salmonella enterica (S. typhi), gil17938413 from Agrobacterium tumefaciens, gil16265654 from Sinorizhobium meliloti, and gil15599932 from Pseudomona aeruginosa. The structure of YjbJ reveals a new variation of a common motif (four-helix bundle) that could not be predicted from the protein sequence. Although the biochemical function is unknown, the existence of patterns of conserved residues on the protein surface suggest that the fold and function of all these proteins could be similar.

A novel member of the split betaalphabeta fold: Solution structure of the hypothetical protein YML108W from Saccharomyces cerevisiae.

As part of the Northeast Structural Genomics Consortium pilot project focused on small eukaryotic proteins and protein domains, we have determined the NMR structure of the protein encoded by ORF YML108W from Saccharomyces cerevisiae. YML108W belongs to one of the numerous structural proteomics targets whose biological function is unknown. Moreover, this protein does not have sequence similarity to any other protein. The NMR structure of YML108W consists of a four‐stranded β‐sheet with strand order 2143 and two α‐helices, with an overall topology of ββαββα. Strand β1 runs parallel to β4, and β2:β1 and β4:β3 pairs are arranged in an antiparallel fashion. Although this fold belongs to the split βαβ family, it appears to be unique among this family; it is a novel arrangement of secondary structure, thereby expanding the universe of protein folds.