Eugene F. DeRose

Crystal Structure of Calmodulin Binding Domain of Orai1 in Complex with Ca2+/Calmodulin Displays a Unique Binding Mode

Background: Calmodulin (CaM) binds the calmodulin binding domain (CMBD) of Orai1 causing Ca2+·CaM-dependent inhibition (CDI). Results: Orai1-CMBD binds the C-lobe of CaM in crystal, whereas in solution Orai1-CMBD interacts with both N- and C-lobes of CaM. Conclusion: One CaM, in an extended conformation, binds to two Orai1-CMBDs. Significance: This expands our knowledge of the interaction of CaM with its targets, and provides important information for understanding CDI. Orai1 is a plasma membrane protein that in its tetrameric form is responsible for calcium influx from the extracellular environment into the cytosol in response to interaction with the Ca2+-depletion sensor STIM1. This is followed by a fast Ca2+·calmodulin (CaM)-dependent inhibition, resulting from CaM binding to an Orai1 region called the calmodulin binding domain (CMBD). The interaction between Orai1 and CaM at the atomic level remains unknown. Here, we report the crystal structure of a CaM·Orai1-CMBD complex showing one CMBD bound to the C-terminal lobe of CaM, differing from other CaM-target protein complexes, in which both N- and C-terminal lobes of CaM (CaM-N and CaM-C) are involved in target binding. Orai1-CMBD binds CaM-C mainly through hydrophobic interactions, primarily involving residue Trp76 of Orai1-CMBD, which interacts with the hydrophobic pocket of CaM-C. However, NMR data, isothermal titration calorimetry data, and pulldown assays indicated that CaM-N and CaM-C both can bind Orai1-CMBD, with CaM-N having ∼4 times weaker affinity than CaM-C. Pulldown assays of a Orai1-CMBD(W76E) mutant, gel filtration chromatography data, and NOE signals indicated that CaM-N and CaM-C can each bind one Orai1-CMBD. Thus our studies support an unusual, extended 1:2 binding mode of CaM to Orai1-CMBDs, and quantify the affinity of Orai1 for CaM. We propose a two-step mechanism for CaM-dependent Orai1 inactivation initiated by binding of the C-lobe of CaM to the CMBD of one Orai1 followed by the binding of the N-lobe of CaM to the CMBD of a neighboring Orai1.

A comparison of BRCT domains involved in nonhomologous end-joining: introducing the solution structure of the BRCT domain of polymerase lambda.

Three of the four family X polymerases, DNA polymerase lambda, DNA polymerase mu, and TdT, have been associated with repair of double-strand DNA breaks by nonhomologous end-joining. Their involvement in this DNA repair process requires an N-terminal BRCT domain that mediates interaction with other protein factors required for recognition and binding of broken DNA ends. Here we present the NMR solution structure of the BRCT domain of DNA polymerase lambda, completing the structural portrait for this family of enzymes. Analysis of the overall fold of the polymerase lambda BRCT domain reveals structural similarity to the BRCT domains of polymerase mu and TdT, yet highlights some key sequence and structural differences that may account for important differences in the biological activities of these enzymes and their roles in nonhomologous end-joining. Mutagenesis studies indicate that the conserved Arg57 residue of Pol lambda plays a more critical role for binding to the XRCC4-Ligase IV complex than its structural homolog in Pol mu, Arg43. In contrast, the hydrophobic Leu60 residue of Pol lambda contributes less significantly to binding than the structurally homologous Phe46 residue of Pol mu. A third leucine residue involved in the binding and activity of Pol mu, is nonconservatively replaced by a glutamine in Pol lambda (Gln64) and, based on binding and activity data, is apparently unimportant for Pol lambda interactions with the NHEJ complex. In conclusion, both the structure of the Pol lambda BRCT domain and its mode of interaction with the other components of the NHEJ complex significantly differ from the two previously studied homologs, Pol mu and TdT.

Structural Studies of the PARP-1 BRCT Domain

BackgroundPoly(ADP-ribose) polymerase-1 (PARP-1) is one of the first proteins localized to foci of DNA damage. Upon activation by encountering nicked DNA, the PARP-1 mediated trans-poly(ADP-ribosyl)ation of DNA binding proteins occurs, facilitating access and accumulation of DNA repair factors. PARP-1 also auto-(ADP-ribosyl)ates its central BRCT-containing domain forming part of an interaction site for the DNA repair scaffolding protein X-ray cross complementing group 1 protein (XRCC1). The co-localization of XRCC1, as well as bound DNA repair factors, to sites of DNA damage is important for cell survival and genomic integrity.ResultsHere we present the solution structure and biophysical characterization of the BRCT domain of rat PARP-1. The PARP-1 BRCT domain has the globular α/β fold characteristic of BRCT domains and has a thermal melting transition of 43.0°C. In contrast to a previous characterization of this domain, we demonstrate that it is monomeric in solution using both gel-filtration chromatography and small-angle X-ray scattering. Additionally, we report that the first BRCT domain of XRCC1 does not interact significantly with the PARP-1 BRCT domain in the absence of ADP-ribosylation. Moreover, none of the interactions with other longer PARP-1 constructs which previously had been demonstrated in a pull-down assay of mammalian cell extracts were detected.ConclusionsThe PARP-1 BRCT domain has the conserved BRCT fold that is known to be an important protein:protein interaction module in DNA repair and cell signalling pathways. Data indicating no significant protein:protein interactions between PARP-1 and XRCC1 likely results from the absence of poly(ADP-ribose) in one or both binding partners, and further implicates a poly(ADP-ribose)-dependent mechanism for localization of XRCC1 to sites of DNA damage.

Preventing oxidation of cellular XRCC1 affects PARP-mediated DNA damage responses.

Poly(ADP-ribose) polymerase-1 (PARP-1) binds intermediates of base excision repair (BER) and becomes activated for poly(ADP-ribose) (PAR) synthesis. PAR mediates recruitment and functions of the key BER factors XRCC1 and DNA polymerase β (pol β) that in turn regulate PAR. Yet, the molecular mechanism and implications of coordination between XRCC1 and pol β in regulating the level of PAR are poorly understood. A complex of PARP-1, XRCC1 and pol β is found in vivo, and it is known that pol β and XRCC1 interact through a redox-sensitive binding interface in the N-terminal domain of XRCC1. We confirmed here that both oxidized and reduced forms of XRCC1 are present in mouse fibroblasts. To further understand the importance of the C12-C20 oxidized form of XRCC1 and the interaction with pol β, we characterized cell lines representing stable transfectants in Xrcc1(-/-) mouse fibroblasts of wild-type XRCC1 and two mutants of XRCC1, a novel reduced form with the C12-C20 disulfide bond blocked (C12A) and a reference mutant that is unable to bind pol β (V88R). XRCC1-deficient mouse fibroblasts are extremely hypersensitive to methyl methanesulfonate (MMS), and transfected wild-type and C12A mutant XRCC1 proteins similarly reversed MMS hypersensitivity. However, after MMS exposure the cellular PAR level was found to increase to a much greater extent in cells expressing the C12A mutant than in cells expressing wild-type XRCC1. PARP inhibition resulted in very strong MMS sensitization in cells expressing wild-type XRCC1, but this sensitization was much less in cells expressing the C12A mutant. The results suggest a role for the oxidized form of XRCC1 in the interaction with pol β in (1) controlling the PAR level after MMS exposure and (2) enabling the extreme cytotoxicity of PARP inhibition during the MMS DNA damage response.

Solution structure of the Drosha double-stranded RNA-binding domain.

BackgroundDrosha is a nuclear RNase III enzyme that initiates processing of regulatory microRNA. Together with partner protein DiGeorge syndrome critical region 8 (DGCR8), it forms the Microprocessor complex, which cleaves precursor transcripts called primary microRNA to produce hairpin precursor microRNA. In addition to two RNase III catalytic domains, Drosha contains a C-terminal double-stranded RNA-binding domain (dsRBD). To gain insight into the function of this domain, we determined the nuclear magnetic resonance (NMR) solution structure.ResultsWe report here the solution structure of the dsRBD from Drosha (Drosha-dsRBD). The αβββα fold is similar to other dsRBD structures. A unique extended loop distinguishes this domain from other dsRBDs of known structure.ConclusionsDespite uncertainties about RNA-binding properties of the Drosha-dsRBD, its structure suggests it retains RNA-binding features. We propose that this domain may contribute to substrate recognition in the Drosha-DGCR8 Microprocessor complex.

Direct Magnetic Resonance Evidence for Peroxymonocarbonate Involvement in the Cu,Zn-Superoxide Dismutase Peroxidase Catalytic Cycle

Cu,Zn-superoxide dismutase (SOD1) is a copper- and zinc-dependent enzyme. The main function of SOD1 is believed to be the scavenging and detoxification of superoxide radicals. Nevertheless, the last 30 years have seen a rapid accumulation of evidence indicating that SOD1 may also act as a peroxidase, an alternative function that was implicated in the onset and progression of familial amyotrophic lateral sclerosis. Although SOD1 peroxidase activity and its dependence on carbon dioxide have been well described, the molecular basis of the SOD1 peroxidase cycle remains obscure, because none of the proposed catalytic intermediates have so far been identified. In view of recent observations, we hypothesized that the SOD1 peroxidase cycle relies on two steps: 1) reduction of SOD-Cu(II) by hydrogen peroxide followed by 2) oxidation of SOD-Cu(I) by peroxymonocarbonate, the product of the spontaneous reaction of bicarbonate with hydrogen peroxide, to produce SOD-Cu(II) and carbonate radical anion. This hypothesis has been investigated through electron paramagnetic resonance and nuclear magnetic resonance to provide direct evidence for a peroxycarbonate-driven, SOD1-catalyzed carbonate radical production. The results gathered herein indicate that peroxymonocarbonate (\batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{HOOCO_{2}^{-}}\) \end{document}) is a key intermediate in the SOD1 peroxidase cycle and identify this species as the precursor of carbonate radical anions.

Structure of the Escherichia coli DNA Polymerase III ϵ-HOT Proofreading Complex

The ϵ subunit of Escherichia coli DNA polymerase III possesses 3′-exonucleolytic proofreading activity. Within the Pol III core, ϵ is tightly bound between the α subunit (DNA polymerase) and θ subunit. Here, we present the crystal structure of ϵ in complex with HOT, the bacteriophage P1-encoded homolog of θ, at 2.1 Å resolution. The ϵ-HOT interface is defined by two areas of contact: an interaction of the previously unstructured N terminus of HOT with an edge of the ϵ central β-sheet as well as interactions between HOT and the catalytically important helix α1-loop-helix α2 motif of ϵ. This structure provides insight into how HOT and, by implication, θ may stabilize the ϵ subunit, thus promoting efficient proofreading during chromosomal replication.

Identification and functional characterization of a novel acetylcholine-binding protein from the marine annelid Capitella teleta.

We identified a homologue of the molluscan acetylcholine-binding protein (AChBP) in the marine polychaete Capitella teleta, from the annelid phylum. The amino acid sequence of C. teleta AChBP (ct-AChBP) is 21-30% identical with those of known molluscan AChBPs. Sequence alignments indicate that ct-AChBP has a shortened Cys loop compared to other Cys loop receptors, and a variation on a conserved Cys loop triad, which is associated with ligand binding in other AChBPs and nicotinic ACh receptor (nAChR) alpha subunits. Within the D loop of ct-AChBP, a conserved aromatic residue (Tyr or Trp) in nAChRs and molluscan AChBPs, which has been implicated directly in ligand binding, is substituted with an isoleucine. Mass spectrometry results indicate that Asn122 and Asn216 of ct-AChBP are glycosylated when expressed using HEK293 cells. Small-angle X-ray scattering data suggest that the overall shape of ct-AChBP in the apo or unliganded state is similar to that of homologues with known pentameric crystal structures. NMR experiments show that acetylcholine, nicotine, and alpha-bungarotoxin bind to ct-AChBP with high affinity, with K(D) values of 28.7 microM, 209 nM, and 110 nM, respectively. Choline bound with a lower affinity (K(D) = 163 microM). Our finding of a functional AChBP in a marine annelid demonstrates that AChBPs may exhibit variations in hallmark motifs such as ligand-binding residues and Cys loop length and shows conclusively that this neurotransmitter binding protein is not limited to the phylum Mollusca.

NMR characterizations of an amyloidogenic conformational ensemble of the PI3K SH3 domain

Amyloid formation is associated with structural changes of native polypeptides to monomeric intermediate states and their self‐assembly into insoluble aggregates. Characterizations of the amyloidogenic intermediate state are, therefore, of great importance in understanding the early stage of amyloidogenesis. Here, we present NMR investigations of the structural and dynamic properties of the acid‐unfolded amyloidogenic intermediate state of the phosphatidylinositol 3‐kinase (PI3K) SH3 domain—a model peptide. The monomeric amyloidogenic state of the SH3 domain studied at pH 2.0 (35°C) was shown to be substantially disordered with no secondary structural preferences. 15N NMR relaxation experiments indicated that the unfolded polypeptide is highly flexible on a subnanosecond timescale when observed under the amyloidogenic condition (pH 2.0, 35°C). However, more restricted motions were detected in residues located primarily in the β‐strands as well as in a loop in the native fold. In addition, nonnative long‐range interactions were observed between the residues with the reduced flexibility by paramagnetic relaxation enhancement (PRE) experiments. These indicate that the acid‐unfolded state of the SH3 domain adopts a partly folded conformation through nonnative long‐range contacts between the dynamically restricted residues at the amyloid‐forming condition.

Selective unfolding of one Ribonuclease H domain of HIV reverse transcriptase is linked to homodimer formation

HIV-1 reverse transcriptase (RT), a critical enzyme of the HIV life cycle and an important drug target, undergoes complex and largely uncharacterized conformational rearrangements that underlie its asymmetric folding, dimerization and subunit-selective ribonuclease H domain (RH) proteolysis. In the present article we have used a combination of NMR spectroscopy, small angle X-ray scattering and X-ray crystallography to characterize the p51 and p66 monomers and the conformational maturation of the p66/p66′ homodimer. The p66 monomer exists as a loosely structured molecule in which the fingers/palm/connection, thumb and RH substructures are connected by flexible (disordered) linking segments. The initially observed homodimer is asymmetric and includes two fully folded RH domains, while exhibiting other conformational features similar to that of the RT heterodimer. The RH′ domain of the p66′ subunit undergoes selective unfolding with time constant ∼6.5 h, consistent with destabilization due to residue transfer to the polymerase′ domain on the p66′ subunit. A simultaneous increase in the intensity of resonances near the random coil positions is characterized by a similar time constant. Consistent with the residue transfer hypothesis, a construct of the isolated RH domain lacking the two N-terminal residues is shown to exhibit reduced stability. These results demonstrate that RH′ unfolding is coupled to homodimer formation.