John A. A. Ladias

Structural Basis of the Na+/H+ Exchanger Regulatory Factor PDZ1 Interaction with the Carboxyl-terminal Region of the Cystic Fibrosis Transmembrane Conductance Regulator

The PDZ1 domain of the Na+/H+ exchanger regulatory factor (NHERF) binds with nanomolar affinity to the carboxyl-terminal sequence QDTRL of the cystic fibrosis transmembrane conductance regulator (CFTR) and plays a central role in the cellular localization and physiological regulation of this chloride channel. The crystal structure of human NHERF PDZ1 bound to the carboxyl-terminal peptide QDTRL has been determined at 1.7-Å resolution. The structure reveals the specificity and affinity determinants of the PDZ1-CFTR interaction and provides insights into carboxyl-terminal leucine recognition by class I PDZ domains. The peptide ligand inserts into the PDZ1 binding pocket forming an additional antiparallel β-strand to the PDZ1 β-sheet, and an extensive network of hydrogen bonds and hydrophobic interactions stabilize the complex. Remarkably, the guanido group of arginine at position −1 of the CFTR peptide forms two salt bridges and two hydrogen bonds with PDZ1 residues Glu43 and Asn22, respectively, providing the structural basis for the contribution of the penultimate amino acid of the peptide ligand to the affinity of the interaction.

Novel Mode of Ligand Recognition by the Erbin PDZ Domain

Erbin contains a class I PDZ domain that binds to the C-terminal region of the receptor tyrosine kinase ErbB2, a class II ligand. The crystal structure of the human Erbin PDZ bound to the peptide EYLGLDVPV corresponding to the C-terminal residues 1247–1255 of human ErbB2 has been determined at 1.25-Å resolution. The Erbin PDZ deviates from the canonical PDZ fold in that it contains a single α-helix. The isopropyl group of valine at position −2 of the ErbB2 peptide interacts with the Erbin Val1351and displaces the peptide backbone away from the α-helix, elucidating the molecular basis of class II ligand recognition by a class I PDZ domain. Strikingly, the phenolic ring of tyrosine −7 enters into a pocket formed by the extended β2-β3 loop of the Erbin PDZ. Phosphorylation of tyrosine −7 abolishes this interaction but does not affect the binding of the four C-terminal peptidic residues to PDZ, as revealed by the crystal structure of the Erbin PDZ complexed with a phosphotyrosine-containing ErbB2 peptide. Since phosphorylation of tyrosine −7 plays a critical role in ErbB2 function, the selective binding and sequestration of this residue in its unphosphorylated state by the Erbin PDZ provides a novel mechanism for regulation of the ErbB2-mediated signaling and oncogenicity.

BRCA1 interaction with RNA polymerase II reveals a role for hRPB2 and hRPB10alpha in activated transcription.

The functions of most of the 12 subunits of the RNA polymerase II (Pol II) enzyme are unknown. In this study, we demonstrate that two of the subunits, hRPB2 and hRPB10alpha, mediate the regulated stimulation of transcription. We find that the transcriptional coactivator BRCA1 interacts directly with the core Pol II complex in vitro. We tested whether single subunits from Pol II would compete with the intact Pol II complex to inhibit transcription stimulated by BRCA1. Excess purified Pol II subunits hRPB2 or hRPB10alpha blocked BRCA1- and VP16-dependent transcriptional activation in vitro with minimal effect on basal transcription. No other Pol II subunits tested inhibited activated transcription in these assays. Furthermore, hRPB10alpha, but not hRPB2, blocked Sp1-dependent activation.

Critical Structural Elements and Multitarget Protein Interactions of the Transcriptional Activator AF-1 of Hepatocyte Nuclear Factor 4

The nuclear receptor hepatocyte nuclear factor 4 (HNF-4) is an important regulator of several genes involved in diverse metabolic and developmental pathways. Mutations in the HNF-4Agene are responsible for the maturity-onset diabetes of the young type 1. Recently, we showed that the 24 N-terminal residues of HNF-4 function as an acidic transcriptional activator, termed AF-1 (Hadzopoulou-Cladaras, M., Kistanova, E., Evagelopoulou, C., Zeng, S., Cladaras C., and Ladias, J. A. A. (1997) J. Biol. Chem. 272, 539–550). To identify the critical residues for this activator, we performed an extensive genetic analysis using site-directed mutagenesis. We showed that the aromatic and bulky hydrophobic residues Tyr6, Tyr14, Phe19, Lys10, and Lys17 are essential for AF-1 function. To a lesser degree, five acidic residues are also important for optimal activity. Positional changes of Tyr6 and Tyr14 reduced AF-1 activity, underscoring the importance of primary structure for this activator. Our analysis also indicated that AF-1 is bipartite, consisting of two modules that synergize to activate transcription. More important, AF-1 shares common structural motifs and molecular targets with the activators of the tumor suppressor protein p53 and NF-κB-p65, suggesting similar mechanisms of action. Remarkably, AF-1 interacted specifically with multiple transcriptional targets, including the TATA-binding protein; the TATA-binding protein-associated factors TAFII31 and TAFII80; transcription factor IIB; transcription factor IIH-p62; and the coactivators cAMP-responsive element-binding protein-binding protein, ADA2, and PC4. The interaction of AF-1 with proteins that regulate distinct steps of transcription may provide a mechanism for synergistic activation of gene expression by AF-1.

Pathogenicity of the BRCA1 missense variant M1775K is determined by the disruption of the BRCT phosphopeptide-binding pocket: a multi-modal approach.

A number of germ-line mutations in the BRCA1 gene confer susceptibility to breast and ovarian cancer. However, it remains difficult to determine whether many single amino-acid (missense) changes in the BRCA1 protein that are frequently detected in the clinical setting are pathologic or not. Here, we used a combination of functional, crystallographic, biophysical, molecular and evolutionary techniques, and classical genetic segregation analysis to demonstrate that the BRCA1 missense variant M1775K is pathogenic. Functional assays in yeast and mammalian cells showed that the BRCA1 BRCT domains carrying the amino-acid change M1775K displayed markedly reduced transcriptional activity, indicating that this variant represents a deleterious mutation. Importantly, the M1775K mutation disrupted the phosphopeptide-binding pocket of the BRCA1 BRCT domains, thereby inhibiting the BRCA1 interaction with the proteins BRIP1 and CtIP, which are involved in DNA damage-induced checkpoint control. These results indicate that the integrity of the BRCT phosphopeptide-binding pocket is critical for the tumor suppression function of BRCA1. Moreover, this study demonstrates that multiple lines of evidence obtained from a combination of functional, structural, molecular and evolutionary techniques, and classical genetic segregation analysis are required to confirm the pathogenicity of rare variants of disease-susceptibility genes and obtain important insights into the underlying pathogenetic mechanisms.

In vitro and in vivo analysis of the binding of the C terminus of the HDL receptor scavenger receptor class B, type I (SR-BI), to the PDZ1 domain of its adaptor protein PDZK1.

The PDZ1 domain of the four PDZ domain-containing protein PDZK1 has been reported to bind the C terminus of the HDL receptor scavenger receptor class B, type I (SR-BI), and to control hepatic SR-BI expression and function. We generated wild-type (WT) and mutant murine PDZ1 domains, the mutants bearing single amino acid substitutions in their carboxylate binding loop (Lys14-Xaa4-Asn19-Tyr-Gly-Phe-Phe-Leu24), and measured their binding affinity for a 7-residue peptide corresponding to the C terminus of SR-BI (503VLQEAKL509). The Y20A and G21Y substitutions abrogated all binding activity. Surprisingly, binding affinities (Kd) of the K14A and F22A mutants were 3.2 and 4.0 μm, respectively, similar to 2.6 μm measured for the WT PDZ1. To understand these findings, we determined the high resolution structure of WT PDZ1 bound to a 5-residue sequence from the C-terminal SR-BI (505QEAKL509) using x-ray crystallography. In addition, we incorporated the K14A and Y20A substitutions into full-length PDZK1 liver-specific transgenes and expressed them in WT and PDZK1 knock-out mice. In WT mice, the transgenes did not alter endogenous hepatic SR-BI protein expression (intracellular distribution or amount) or lipoprotein metabolism (total plasma cholesterol, lipoprotein size distribution). In PDZK1 knock-out mice, as expected, the K14A mutant behaved like wild-type PDZK1 and completely corrected their hepatic SR-BI and plasma lipoprotein abnormalities. Unexpectedly, the 10–20-fold overexpressed Y20A mutant also substantially, but not completely, corrected these abnormalities. The results suggest that there may be an additional site(s) within PDZK1 that bind(s) SR-BI and mediate(s) productive SR-BI-PDZK1 interaction previously attributed exclusively to the canonical binding of the C-terminal SR-BI to PDZ1.

Solution Structure of the Hrpabc14.4 Subunit of Human RNA Polymerases

The protein hRPABC14.4 is an essential subunit of human RNA polymerases I, II, and III and is required for the transcription of all human nuclear genes. The structure of hRPABC14.4 was determined by nuclear magnetic resonance spectroscopy. The protein fold comprises a highly conserved central domain forming two antiparallel α-helices flanked by the less conserved N- and C-terminal regions forming a five-stranded β-sandwich. Amino acids from the two helices participate in the generation of a hydrophobic surface area which is conserved in all eukaryotic and archaeal homologous subunits, and likely constitutes a critical macromolecular interaction interface. The hRPABC14.4 structure accounts for mutagenesis results in Saccharomyces cerevisiae and provides a structural working model for elucidating the role of this subunit in the molecular architecture and function of the human nuclear RNA polymerases.

Genetic linkage of the human apolipoprotein AI-CIII-AIV gene cluster and the neural cell adhesion molecule (NCAM) gene

The genes encoding apolipoproteins AI, CIII, and AIV, three plasma proteins involved in lipid metabolism, are clustered within a 15-kb DNA segment (apoAI-CIII-AIV gene cluster) located on human chromosome 11 at band q23. The gene encoding the neural cell adhesion molecule (NCAM), a cell surface glycoprotein involved in cell-cell recognition during morphogenesis, is also located on chromosome 11, band q23. In this report, 12 previously described restriction fragment length polymorphisms (RFLPs) in the apoAI-CIII-AIV gene cluster were tested for cosegregation with a newly identified BamHI RFLP in the NCAM gene using 13 families. The results show that the apoAI-CIII-AIV gene cluster and the NCAM gene loci are linked with a maximum lod score of 15.9 at a recombination fraction of 0.028. In addition, an approach for the most efficient use of the apoAI-CIII-AIV gene cluster polymorphisms, based on the evaluation of their individual and cumulative heterozygosities, is presented.

Solution structure of the human Tax-interacting protein-1.

PDZ (PSD95/DLG/ZO-1) domains are protein–protein interaction modules composed of 90–100 amino acids. PDZ-containing proteins function in the organization of multiprotein complexes at specific cellular sites and control the spatial and temporal fidelity of intracellular signaling pathways. The PDZ fold comprises a compact six-stranded b-barrel (b1–b6) capped by two a-helices (a1–a2) (Doyle et al. 1996). Target protein recognition is accomplished via a long and deep groove formed between b2 and a2. The C terminus of target proteins inserts into this groove and augments the b-sheet structure of the domain. Certain PDZ domains interact with internal sequence motifs, whereas others bind lipids. Previous studies have established three PDZ classes based on their consensus recognition sequence. Class I prefer X-S/T-X-U-COOH, class II interact with X-U-X-U-COOH, and class III recognize X-D/E-X-U-COOH, where X is any amino acid and U is a hydrophobic residue. However, many PDZ domains bind promiscuously to targets from different classes, underscoring the need for a new classification scheme (Birrane et al. 2003). In general, PDZ proteins possess multiple domains facilitating their functions as protein–protein interaction modules in a variety of cellular contexts. The human Taxinteracting protein-1 (TIP-1) is an unusual PDZ protein predicted to be comprised entirely of a single PDZ domain. TIP-1 was initially identified as an interaction partner of the human T-lymphotropic virus type 1 (HTLV-1) Tax protein, a key viral regulatory protein involved in deregulating numerous cellular signal transduction pathways (Rousset et al. 1998). TIP-1 was also independently identified as a protein that associates with glutaminase L and was termed glutaminase-interacting protein (Olalla et al. 2001). TIP-1 participates in Rho signaling through its interaction with Rhotekin (Reynaud et al. 2000). In addition, TIP-1 binds with high affinity to the C terminus of b-catenin and inhibits its transcriptional activity (Kanamori et al. 2003). TIP-1 also interacts with the C-terminal tail of the Kir 2.3 potassium channel and functions as a negative regulator of its surface expression (Alewine et al. 2006). As a first step towards understanding the structural basis of TIP-1 interaction with cellular and viral proteins, we determined the solution structure of TIP-1 in the apo form and studied its interaction with a Kir 2.3 C-terminal peptide, using nuclear magnetic resonance (NMR) spectroscopy. We show that TIP-1 consists of a single PDZ domain flanked by unstructured N and C termini. Unique features of the TIP-1 PDZ domain include a short, two-stranded b-sheet within its b1–b2 loop, and a long b2–b3 loop that may participate in ligand binding. Extensive chemical shift perturbations are observed during titration of the protein with a Kir 2.3 peptide, indicating that TIP-1 undergoes conformational changes upon interaction with target proteins. M. A. Durney G. Birrane A. Soni J. A. A. Ladias (&) Molecular Medicine Laboratory and Macromolecular Crystallography Unit, Department of Medicine, Harvard Medical School, Boston, MA 02215, USA e-mail: johnladias@gmail.com

Structural Basis for the BRCA1 BRCT Interaction with the Proteins ATRIP and BAAT1.

The breast and ovarian cancer susceptibility protein 1 (BRCA1) plays a central role in DNA damage response (DDR). Two tandem BRCA1 C-terminal (BRCT) domains interact with several proteins that function in DDR and contain the generally accepted motif pS-X-X-F (pS denoting phosphoserine and X any amino acid), including the ATR-interacting protein (ATRIP) and the BRCA1-associated protein required for ATM activation-1 (BAAT1). The crystal structures of the BRCA1 BRCTs bound to the phosphopeptides ATRIP (235-PEACpSPQFG-243) and BAAT1 (266-VARpSPVFSS-274) were determined at 1.75 Å and 2.2 Å resolution, respectively. The pSer and Phe(+3) anchor the phosphopeptides into the BRCT binding groove, with adjacent peptide residues contributing to the interaction. In the BRCA1-ATRIP structure, Gln(+2) is accommodated through a conformational change of the BRCA1 E1698 side chain. Importantly, isothermal titration calorimetry experiments showed that the size and charge of the side chains at peptide positions +1 and +2 contribute significantly to the BRCA1 BRCT-peptide binding affinity. In particular, the Asp(+1) and Glu(+2) in the human CDC27 peptide 816-HAAEpSDEF-823 abrogate the interaction with the BRCA1 BRCTs due in large part to electrostatic repulsion between Glu(+2) and E1698, indicating a preference of these domains for specific side chains at positions +1 and +2. These results emphasize the need for a systematic assessment of the contribution of the peptide residues surrounding pSer and Phe(+3) to the binding affinity and specificity of the BRCA1 BRCTs in order to elucidate the molecular mechanisms underlying the hierarchy of target selection by these versatile domains during DDR and tumorigenesis.