Jan Aelen

Ca2+ regulation in the Na+/Ca2+ exchanger features a dual electrostatic switch mechanism

Regulation of ion-transport in the Na+/Ca2+ exchanger (NCX) occurs via its cytoplasmic Ca2+-binding domains, CBD1 and CBD2. Here, we present a mechanism for NCX activation and inactivation based on data obtained using NMR, isothermal titration calorimetry (ITC) and small-angle X-ray scattering (SAXS). We initially determined the structure of the Ca2+-free form of CBD2-AD and the structure of CBD2-BD that represent the two major splice variant classes in NCX1. Although the apo-form of CBD2-AD displays partially disordered Ca2+-binding sites, those of CBD2-BD are entirely unstructured even in an excess of Ca2+. Striking differences in the electrostatic potential between the Ca2+-bound and -free forms strongly suggest that Ca2+-binding sites in CBD1 and CBD2 form electrostatic switches analogous to C2-domains. SAXS analysis of a construct containing CBD1 and CBD2 reveals a conformational change mediated by Ca2+-binding to CBD1. We propose that the electrostatic switch in CBD1 and the associated conformational change are necessary for exchanger activation. The response of the CBD1 switch to intracellular Ca2+ is influenced by the closely located cassette exons. We further propose that Ca2+-binding to CBD2 induces a second electrostatic switch, required to alleviate Na+-dependent inactivation of Na+/Ca2+ exchange. In contrast to CBD1, the electrostatic switch in CBD2 is isoform- and splice variant-specific and allows for tailored exchange activities.

Association of nuclear DNA with a rapidly sedimenting structure

Abstract Nuclei were isolated from monolayer cultures of bovine liver cells by use of a Triton containing Tris buffer, and dissolved in 1 M NaCl. Analysis of the lysate by sedimentation through sucrose gradients revealed that a variable proportion of the DNA, sometimes as much as 90%, remained associated with a rapidly sedimenting structure. The rapidly sedimenting material was degraded to smaller fragments by pronase digestion, indicating that proteins are essential to maintain the structural properties conferring the high rate of sedimentation. DNA could be released almost quantitatively by DNAase digestion without causing a significant change of the sedimentation rate of the supporting structure. A limited digestion showed that newly synthesized DNA was more resistant against release from the support. It is suggested that DNA molecules are attached to the rapidly sedimenting structure by one binding site per replicon, and that during replication additional attachment sites are generated by DNA regions at the replication forks.

Structural basis for Ca2+ regulation in the Na+/Ca2+ exchanger.

Abstract:  Binding of Na+ and Ca2+ ions to the large cytosolic loop of the Na+/Ca2+ exchanger (NCX) regulates its ion transport across the plasma membrane. We determined the solution structures of two Ca2+‐binding domains (CBD1 and CBD2) that, together with an α‐catenin‐like domain (CLD) form the regulatory exchanger loop. CBD1 and CBD2 constitute a novel Ca2+‐binding motif and are very similar in the Ca2+‐bound state. Strikingly, in the absence of Ca2+ the upper half of CBD1 unfolds while CBD2 maintains its structural integrity. Together with a sevenfold higher affinity for Ca2+ this suggests that CBD1 is the primary Ca2+ sensor. Specific point mutations in either domain largely allow the interchange of their functionality and uncover the mechanism underlying Ca2+ sensing in NCX.

The interaction of PTP-BL PDZ domains with RIL: An enigmatic role for the RIL LIM domain

PDZ domains are protein-protein interaction modules that are crucial for the assembly of structural and signaling complexes. PDZ domains specifically bind short carboxyl-terminal peptides and occasionally internal sequences that structurally resemble peptide termini. Previously, using yeast two-hybrid methodology, we studied the interaction of two PDZ domains present in the large submembranous protein tyrosine phosphatase PTP-BL with the C-terminal half of the LIM domain-containing protein RIL. Deletion of the extreme RIL C-terminus did not eliminate binding, suggesting the presence of a PDZ binding site within the RIL LIM moiety. We have now performed experiments in mammalian cell lysates and found that the RIL C-terminus proper, but not the RIL LIM domain, can interact with PTP-BL, albeit very weakly. However, this interaction with PTP-BL PDZ domains is greatly enhanced when the combined RIL LIM domain and C-terminus is used, pointing to synergistic effects. NMR titration experiments and site-directed mutagenesis indicate that this result is not dependent on specific interactions that require surface exposed residues on the RIL LIM domain, suggesting a stabilizing role in the association with PTP-BL.

Letter to the Editor: Sequence-specific assignment of the PAH2 domain of Sin3B free and bound to Mad1

Key words: assignment, Mad1, PAH domain, Sin3Biological contextThe evolutionary conserved protein Sin3 mediates theassembly of a large multi-protein complex involved inmodification of the local chromatin structure. Sin3 hasbeen shown to interact with a number of proteins, suchas HDAC, N-CoR, Sap30 and Mad1. Sin3 comprisesfour repeats of approximately 80 amino acids, calledPaired Amphipathic-Helix (PAH) domains, that me-diate the interactions of Sin3 with its partners. Thesecond PAH domain of Sin3A and -B has been shownto specifically interact with the N-terminus of Mad1(Mad1-SID, Sin3Interaction Domain) (Ayer et al.,1995; Eilers et al., 1999; Xu et al., 1999), a DNA-binding protein that antagonizes the transcriptionalactivation, proliferation-promotingand transformationfunctions of the onco-protein Myc (Schreiber-Aguset al., 1998; Foley et al., 1999).Methods and experimentsThe fragment containing the PAH2 domain (residues148–252) of mouse Sin3B was obtained by PCR andclonedinto pGEX2T.Using site-directedmutagenesis,the C241A mutation was introduced in the PAH2 do-main to prevent disulphide-bridgemediated dimer for-mation in solution. The resulting GST-PAH2(C241A)fusion protein was expressed in E. coli strain pBL21using 2YT medium supplemented with 0.5% glu-cose. Minimal medium was supplemented with

Newly-initiated DNA isolated from Physarum in early S phase consists of nascent-nascent duplexes.

We have studied the nature of newly initiated DNA released during DNA isolation at the beginning of S phase of Physarum polycephalum. The released DNA was separated from the bulk DNA by sedimentation through sucrose gradients. Gentle shearing strongly enhanced the release of newly initiated DNA. The additionally released material had a larger average molecular weight. Buoyant density analysis after labelling with bromodeoxyuridine (BrdU) revealed that the released DNA consisted of nascent-nascent duplexes for more than 90%. This indicates that the release of newly initiated DNA occurs by branch migration. We conclude that shearing enhances branch migration by destabilization of the double helix.