Nancy Walker-Kopp

Phospholipid Scramblase 1 Contains a Nonclassical Nuclear Localization Signal with Unique Binding Site in Importin α

Nuclear import of proteins containing a classical nuclear localization signal (NLS) is an energy-dependent process that requires the heterodimer importin α/β. Three to six basic contiguous arginine/lysine residues characterize a classical NLS and are thought to form a basic patch on the surface of the import cargo. In this study, we have characterized the NLS of phospholipid scramblase 1 (PLSCR1), a lipid-binding protein that enters the nucleus via the nonclassical NLS 257GKISKHWTGI266. This import sequence lacks a contiguous stretch of positively charged residues, and it is enriched in hydrophobic residues. We have determined the 2.2 Å crystal structure of a complex between the PLSCR1 NLS and the armadillo repeat core of vertebrate importin α. Our crystallographic analysis reveals that PLSCR1 NLS binds to armadillo repeats 1–4 of importin α, but its interaction partially overlaps the classical NLS binding site. Two PLSCR1 lysines occupy the canonical positions indicated as P2 and P5. Moreover, we present in vivo evidence that the critical lysine at position P2, which is essential in other known NLS sequences, is dispensable in PLSCR1 NLS. Taken together, these data provide insight into a novel nuclear localization signal that presents a distinct motif for binding to importin α.

Molecular basis for the recognition of snurportin 1 by importin beta.

The nuclear import of uridine-rich ribonucleoproteins is mediated by the transport adaptor snurportin 1 (SNP1). Similar to importin α, SNP1 uses an N-terminal importin β binding (sIBB) domain to recruit the receptor importin β and gain access to the nucleus. In this study, we demonstrate that the sIBB domain has a bipartite nature, which contains two distinct binding determinants for importin β. The first determinant spans residues 25-65 and includes the previously identified importin α IBB (αIBB) region of homology. The second binding determinant encompasses residues 1-24 and resembles region 1011-1035 of the nucleoporin 153 (Nup153). The two binding determinants synergize within the sIBB domain to confer a low nanomolar binding affinity for importin β (Kd ∼ 2 nm) in an interaction that, in vitro, is displaced by RanGTP. We propose that in vivo the synergy of Nup153 and nuclear RanGTP promotes translocation of uridine-rich ribonucleoproteins into the nucleus.

Foldon‐guided self‐assembly of ultra‐stable protein fibers

A common objective in protein engineering is the enhancement of the thermodynamic properties of recombinant proteins for possible applications in nanobiotechnology. The performance of proteins can be improved by the rational design of chimeras that contain structural elements with the desired properties, thus resulting in a more effective exploitation of protein folds designed by nature. In this paper, we report the design and characterization of an ultra‐stable self‐refolding protein fiber, which rapidly reassembles in solution after denaturation induced by harsh chemical treatment or high temperature. This engineered protein fiber was constructed on the molecular framework of bacteriophage P22 tail needle gp26, by fusing its helical core to the foldon domain of phage T4 fibritin. Using protein engineering, we rationally permuted the foldon upstream and downstream from the gp26 helical core and characterized gp26‐foldon chimeras by biophysical analysis. Our data demonstrate that one specific protein chimera containing the foldon immediately downstream from the gp26 helical core, gp26(1‐140)‐F, displays the highest thermodynamic and structural stability and refolds spontaneously in solution following denaturation. The gp26‐foldon chimeric fiber remains stable in 6.0 M guanidine hydrochloride, or at 80°C, rapidly refolds after denaturation, and has both N and C termini accessible for chemical/biological modification, thereby representing an ideal platform for the design of self‐assembling nanoblocks.

Conserved ETS Domain Arginines Mediate DNA Binding, Nuclear Localization, and a Novel Mode of bZIP Interaction

The DNA-binding ETS transcription factor Spi-1/PU.1 is of central importance in determining the myeloid-erythroid developmental switch and is required for monocyte and osteoclast differentiation. Many monocyte genes are dependent upon this factor, including the gene that codes for interleukin-1β. It has long been known that the conserved ETS DNA-binding domain of Spi-1/PU.1 functionally cooperates via direct association with a diverse collection of DNA-binding proteins, including members of the basic leucine zipper domain (bZIP) family. However, the molecular basis for this interaction has long been elusive. Using a combination of approaches, we have mapped a single residue on the surface of the ETS domain critical for protein tethering by the C/EBPβ carboxylterminal bZIP domain. This residue is also important for nuclear localization and DNA binding. In addition, dependence upon the leucine zipper suggests a novel mode for both protein-DNA interaction and functional cooperativity.

An Evolutionarily Conserved Family of Virion Tail Needles Related to Bacteriophage P22 gp26: Correlation between Structural Stability and Length of the α-Helical Trimeric Coiled Coil

Bacteriophages of the Podoviridae family use short noncontractile tails to inject their genetic material into Gram-negative bacteria. In phage P22, the tail contains a thin needle, encoded by the phage gene 26, which is essential both for stabilization and for ejection of the packaged viral genome. Bioinformatic analysis of the N-terminal domain of gp26 (residues 1-60) led us to identify a family of genes encoding putative homologues of the tail needle gp26. To validate this idea experimentally and to explore their diversity, we cloned the gp26-like gene from phages HK620, Sf6 and HS1, and characterized these gene products in solution. All gp26-like factors contain an elongated alpha-helical coiled-coil core consisting of repeating, adjacent trimerization heptads and form trimeric fibers with length ranging between about 240 to 300 A. gp26 tail needles display a high level of structural stability in solution, with T(m) (temperature of melting) between 85 and 95 degrees C. To determine how the structural stability of these phage fibers correlates with the length of the alpha-helical core, we investigated the effect of insertions and deletions in the helical core. In the P22 tail needle, we identified an 85-residue-long helical domain, termed MiCRU (minimal coiled-coil repeat unit), that can be inserted in-frame inside the gp26 helical core, preserving the straight morphology of the fiber. Likewise, we were able to remove three quarters of the helical core of the HS1 tail needle, minimally decreasing the stability of the fiber. We conclude that in the gp26 family of tail needles, structural stability increases nonlinearly with the length of the alpha-helical core. Thus, the overall stability of these bacteriophage fibers is not solely dependent on the number of trimerization repeats in the alpha-helical core.

Inhibition of IL-1β Transcription by Peptides Derived from the hCMV IE2 Transactivator

The immediate early (IE) proteins of human cytomegalovirus (hCMV) have diverse roles in directing viral and host cell transcription. Among these is the ability of IE2 to induce transcription of the IL1B gene that codes for IL-1beta in monocytes. This function is partially explained by interaction between IE2 and the host cell transcription factor Spi-1/PU.1 (Spi-1). We now show that maximal IE2 function also depends on productive interactions localizing to two C/EBP sites on the IL1B promoter suggesting either bi- or tri-molecular interactions between IE2, Spi-1 and C/EBPbeta at two different locations on the promoter. The IE2 interaction region on Spi-1 was previously mapped to the DNA-binding ETS domain and overlaps the region of Spi-1 that interacts with the transcription factor C/EBPbeta, a factor known to be critical for the induction of IL1B in response to Toll/IL-1 receptor (TIR) family signal transduction. The Spi-1 interacting region of IE2 maps to amino acids 315-328, a sequence that also interacts with the bZIP domain of C/EBPbeta. An expression vector coding for amino acids 291-364 of IE2 can suppress LPS induction of a co-transfected IL1B enhancer-promoter fragment in a monocyte cell line. This inhibition is likely the result of competition between Spi-1 and C/EBPbeta, thus blunting gene induction.

Treacher Collins syndrome mutations in Saccharomyces cerevisiae destabilize RNA polymerase I and III complex integrity

Treacher Collins syndrome (TCS) is a craniofacial disorder that is characterized by the malformation of the facial bones. Mutations in three genes (TCOF1, POLR1C and POLR1D) involved in RNA polymerase I (Pol I) transcription account for more than 90% of disease cases. Two of these TCS-associated genes, POLR1C and POLR1D, encode for essential Pol I/III subunits that form a heterodimer necessary for Pol I/III assembly, and many TCS mutations lie along their evolutionarily conserved dimerization interface. Here we elucidate the molecular basis of TCS mutations in Saccharomyces cerevisiae, and present a new model for how TCS mutations may disrupt Pol I and III complex integrity.

Reconstitution of RNA Polymerase I Upstream Activating Factor and the Roles of Histones H3 and H4 in Complex Assembly

RNA polymerase I (Pol I) transcription in Saccharomyces cerevisiae requires four separate factors that recruit Pol I to the promoter to form a pre-initiation complex. Upstream Activating Factor (UAF) is one of two multi-subunit complexes that regulate pre-initiation complex formation by binding to the ribosomal DNA promoter and by stimulating recruitment of downstream Pol I factors. UAF is composed of Rrn9, Rrn5, Rrn10, Uaf30, and histones H3 and H4. We developed a recombinant Escherichia coli-based system to coexpress and purify transcriptionally active UAF complex and to investigate the importance of each subunit in complex formation. We found that no single subunit is required for UAF assembly, including histones H3 and H4. We also demonstrate that histone H3 is able to interact with each UAF-specific subunit, and show that there are at least two copies of histone H3 and one copy of H4 present in the complex. Together, our results provide a new model suggesting that UAF contains a hybrid H3-H4 tetramer-like subcomplex.

Super elongation complex contains a TFIIF-related subcomplex

ABSTRACT Super elongation complex (SEC) belongs to a family of RNA polymerase II (Pol II) elongation factors that has similar properties as TFIIF, a general transcription factor that increases the transcription elongation rate by reducing pausing. Although SEC has TFIIF-like functional properties, it apparently lacks sequence and structural homology. Using HHpred, we find that SEC contains an evolutionarily related TFIIF-like subcomplex. We show that the SEC subunit ELL interacts with the Pol II Rbp2 subunit, as expected for a TFIIF-like factor. These findings suggest a new model for how SEC functions as a Pol II elongation factor and how it suppresses Pol II pausing.