Long chain and short chain curaremimetic toxins from snakes possess 66 -74 residues with five disulfide bonds and 60 -62 residues with four disulfide bonds, respectively. Despite their structural differences all of these toxins bind with high affinity to the peripheral nicotinic acetylcholine receptors (AChR). Binding experiments have now revealed that long chain toxins only, like the neuronal -bungarotoxin, have a high affinity for a chimeric form of the neuronal ␣7 receptor, with K d values ranging from about 1 to 12 nM. In contrast, all other toxins bind to the chimeric ␣7 receptor with a low affinity, with K d values ranging between 3 and 22 M. These results are supported by electrophysiological recordings on both the wild-type and chimeric ␣7 receptors. Amino acid sequence analyses have suggested that high affinities for the neuronal receptor are associated with the presence of the fifth disulfide at the tip of the toxin second loop. In agreement with this conclusion, we show that a long chain toxin whose fifth disulfide is reduced and then dithiopyridylated has a low affinity (K d ‫؍‬ 12 M) for the neuronal ␣7 receptor, whereas it retains a high affinity (K d ‫؍‬ 0.35 nM) for the peripheral AChR. Thus, a long chain curaremimetic toxin having a reduced fifth disulfide bond behaves like a short chain toxin toward both the peripheral and neuronal AChR. Therefore, functional classification of toxins that bind to AChRs should probably be done by considering their activities on both peripheral and neuronal receptors.

Cernunnos/XLF is a core protein of the nonhomologous DNA end-joining (NHEJ) pathway that processes the majority of DNA double-strand breaks in mammals. Cernunnos stimulates the final ligation step catalyzed by the complex between DNA ligase IV and Xrcc4 (X4). Here we present the crystal structure of the X4 1-157 -Cernunnos 1-224 complex at 5.5-Å resolution and identify the relative positions of the two factors and their binding sites. The X-ray structure reveals a filament arrangement for X4 1-157 and Cernunnos 1-224 homodimers mediated by repeated interactions through their N-terminal head domains. A filament arrangement of the X4-Cernunnos complex was confirmed by transmission electron microscopy analyses both with truncated and full-length proteins. We further modeled the interface and used structure-based site-directed mutagenesis and calorimetry to characterize the roles of various residues at the X4-Cernunnos interface. We identified four X4 residues (Glu 55 , Asp 58 , Met 61 , and Phe 106 ) essential for the interaction with Cernunnos. These findings provide new insights into the molecular bases for stimulatory and bridging roles of Cernunnos in the final DNA ligation step.D NA double-strand breaks (DSBs) are the most toxic DNA lesions in the genome, and unrepaired DSBs can cause large-scale losses of genetic information through chromosome rearrangement (1, 2). These DNA damages result from exposure to exogenous damaging agents, such as ionizing radiation, radiomimetic compounds, and topoisomerase inhibitors. DSBs are also obligate intermediates in several recombination processes in vertebrates, including antigen receptor gene rearrangement, V(D)J recombination (3, 4). In higher eukaryotes, DSBs are repaired by several mechanisms, among which nonhomologous end-joining (NHEJ) represents the major pathway, particularly when sister chromatids are not available (5). Deficiency in the NHEJ machinery results in sensitivity to ionizing radiation and severe combined immune deficiencies in humans and mice due to abortive V(D)J recombination (6). NHEJ is orchestrated by at least seven proteins. The Ku70/Ku80 heterodimer adopts a preformed ring-shaped structure that recognizes and encircles the duplex DNA ends at the DSB (7). Ku70/Ku80 recruits a 469-kDa serine/threonine protein kinase, the DNA-PK catalytic subunit (DNA-PKcs), via a direct interaction and shifts about 10 bp inward so that DNA-PKcs acquires a position at the terminus through its large open-ring cradle structure (8). Upon association with Ku and DNA, DNA-PKcs is activated and phosphorylates several proteins including itself and Ku70/Ku80. The DNA-PK holoenzyme, constituted by Ku and DNA-PKcs, plays a central role in NHEJ. Among other functions, DNA-PK mediates the end-bridging of the DSB extremities (9), regulates access to the DNA ends by processing enzymes such as the DNA-PKcs-associated Artemis nuclease (10, 11), and recruits the Xrcc4-ligase IV complex to DNA ends for the ligation step (12). The Xrcc4-ligase IV complex carries out the final joinin...

The Ku70-Ku80 (Ku) heterodimer binds rapidly and tightly to ends of DNA double-strand breaks and recruits several factors of the Non-Homologous End Joining (NHEJ) pathway through molecular mechanisms that remain unclear. Here, we describe the crystal structures of the Ku-binding motifs (KBM) of the NHEJ proteins APLF (A-KBM) and XLF (X-KBM) bound to a Ku-DNA complex. The two KBMs motifs bind on remote sites of Ku80 α/β domain. The X-KBM occupies an internal pocket formed after an unprecedented large outward rotation of the Ku80 α/β domain. We reveal independent recruitment at laser-irradiated sites of the APLF-interacting protein XRCC4 and of XLF through the respective binding of A- and X-KBMs to Ku80. Finally, we show that mutations on the X-KBM and A KBM binding sites in Ku80 compromises efficiency and accuracy of end-joining and cellular radiosensitivity. A- and X-KBMs may represent two initial anchorage points necessary to build the NHEJ intricate interactions network.