Ubiquitin-specific proteases (USPs) reverse ubiquitination and regulate virtually all cellular processes. Defined noncatalytic domains in USP4 and USP15 are known to interact with E3 ligases and ...substrate recruitment factors. No such interactions have been reported for these domains in the paralog USP11, a key regulator of DNA double-strand break repair by homologous recombination. We hypothesized that USP11 domains adjacent to its protease domain harbor unique peptide-binding sites. Here, using a next-generation phage display (NGPD) strategy, combining phage display library screening with next-generation sequencing, we discovered unique USP11-interacting peptide motifs. Isothermal titration calorimetry disclosed that the highest affinity peptides (KD of ∼10 μm) exhibit exclusive selectivity for USP11 over USP4 and USP15 in vitro. Furthermore, a crystal structure of a USP11–peptide complex revealed a previously unknown binding site in USP11’s noncatalytic ubiquitin-like (UBL) region. This site interacted with a helical motif and is absent in USP4 and USP15. Reporter assays using USP11-WT versus a binding pocket–deficient double mutant disclosed that this binding site modulates USP11’s function in homologous recombination–mediated DNA repair. The highest affinity USP11 peptide binder fused to a cellular delivery sequence induced significant nuclear localization and cell cycle arrest in S phase, affecting the viability of different mammalian cell lines. The USP11 peptide ligands and the paralog-specific functional site in USP11 identified here provide a framework for the development of new biochemical tools and therapeutic agents. We propose that an NGPD-based strategy for identifying interacting peptides may be applied also to other cellular targets.
The AAA (ATPase associated with various cellular activities) ATPase, p97, is a hexameric protein of chaperone-like function, which has been reported to interact with a number of proteins of seemingly ...unrelated functions. For the first time, we report a classification of these proteins and aim to elucidate any common structural or functional features they may share. The interactors are grouped into those containing ubiquitin regulatory X domains, which presumably bind to p97 in the same way as the p47 adaptor, and into non-ubiquitin regulatory X domain proteins of different functional subgroups that may employ a different mode of interaction (assuming they also bind directly to p97 and are not experimental artifacts). Future studies will show whether interacting proteins direct p97 to different cellular pathways or a common one and structural elucidation of these interactions will be crucial in understanding these underlying functions.
p47 is a major adaptor molecule of the cytosolic AAA ATPase p97. The principal role of the p97–p47 complex is in regulation of membrane fusion events. Mono‐ubiquitin recognition by p47 has also been ...shown to be crucial in the p97–p47‐mediated Golgi membrane fusion events. Here, we describe the high‐resolution solution structures of the N‐terminal UBA domain and the central domain (SEP) from p47. The p47 UBA domain has the characteristic three‐helix bundle fold and forms a highly stable complex with ubiquitin. We report the interaction surfaces of the two proteins and present a structure for the p47 UBA–ubiquitin complex. The p47 SEP domain adopts a novel fold with a βββααβ secondary structure arrangement, where β4 pairs in a parallel fashion to β1. Based on biophysical studies, we demonstrate a clear propensity for the self‐association of p47. Furthermore, p97 N binding abolishes p47 self‐association, revealing the potential interaction surfaces for recognition of other domains within p97 or the substrate.
Coagulation factor XII (FXII) is a key initiator of the contact pathway, which contributes to inflammatory pathways. FXII circulates as a zymogen, which when auto‐activated forms factor XIIa (FXIIa). ...Here, the production of the recombinant FXIIa protease domain (βFXIIaHis) with yields of ∼1–2 mg per litre of insect‐cell culture is reported. A second construct utilized an N‐terminal maltose‐binding protein (MBP) fusion (MBP‐βFXIIaHis). Crystal structures were determined of MBP‐βFXIIaHis in complex with the inhibitor d‐Phe‐Pro‐Arg chloromethyl ketone (PPACK) and of βFXIIaHis in isolation. The βFXIIaHis structure revealed that the S2 and S1 pockets were occupied by Thr and Arg residues, respectively, from an adjacent molecule in the crystal. The Thr‐Arg sequence mimics the P2–P1 FXIIa cleavage‐site residues present in the natural substrates prekallikrein and FXII, and Pro‐Arg (from PPACK) mimics the factor XI cleavage site. A comparison of the βFXIIaHis structure with the available crystal structure of the zymogen‐like FXII protease revealed large conformational changes centred around the S1 pocket and an alternate conformation for the 99‐loop, Tyr99 and the S2 pocket. Further comparison with activated protease structures of factors IXa and Xa, which also have the Tyr99 residue, reveals that a more open form of the S2 pocket only occurs in the presence of a substrate mimetic. The FXIIa inhibitors EcTI and infestin‐4 have Pro‐Arg and Phe‐Arg P2–P1 sequences, respectively, and the interactions that these inhibitors make with βFXIIa are also described. These structural studies of βFXIIa provide insight into substrate and inhibitor recognition and establish a scaffold for the structure‐guided drug design of novel antithrombotic and anti‐inflammatory agents.
Coagulation factor XII (FXII) is a key initiator of the contact pathway and of kinin generation. Here, the first peptidomimetic complex structure of the activated protease domain βFXIIa is reported. The crystal structures of βFXIIa provide insight into substrate and inhibitor recognition by serine proteases.
The principle of ligand exchange has been applied to the enantioseparation of underivatized aromatic and aliphatic amino acids as well as dipeptides. Two non commercially available
N-alkyl-
...l-4-hydroxyproline derivatives were compared to underivatized
l-4-hydroxyproline for their ability to resolve α-amino acids and dipeptides.
N-(2-hydroxyoctyl)-
l-4-hydroxyproline and
N-(2-hydroxypropyl)-
l-4-hydroxyproline were used as their copper(II) complexes as chiral selectors. With these selectors, several aliphatic amino acids and dipeptides, in addition to aromatic amino acids, were resolved. The pH optimum was found to be 4.3 for amino acids and 6.0 for dipeptides.
The development of a competitive solid-phase immunoassay for digoxin making use of the acridinium chemiluminescence system is described. Two different instrumental approaches are compared. One is ...based on a continuous flow system using a peristaltic flow injection analysis pump; the other uses a new sequential injection technique. In both systems a flow cell, consisting of transparent PTFE tubing packed with immobilized antibodies, acts as an immunoreactor. The entire assay, including both the immunoreaction and the chemiluminescence reaction, takes place in this immunoreactor cell. Compared with the flow injection technique, the sequential injection mode showed higher precision, ranging from 2.16 to 5.5% RSD depending on concentration. The total assay time, including regeneration, is less than 8 min with the sequential injection technique. The detection limit for both techniques is in the low femtomole range.
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