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Organized by unstructured motifs The high degree of conservation in protein sequences thought to be unstructured has hinted that these regions may have important biological functions. Although unstructured regions are widely viewed to be crucial for protein signaling, localization, and stability, their roles in many other settings have remained mysterious. Cermakova et al . discovered that prominent members of the transcription elongation machinery are linked through a network of interactions involving transcription elongation factor TFIIS N-terminal domains (TNDs) and conserved unstructured sequences called “TND-interacting motifs” (TIMs). The researchers found that mutation of a single TIM in a central organizing protein of this network abolished key protein interactions and induced widespread defects in transcription elongation dynamics. —DJ

Dimerization of many eukaryotic transcription regulatory factors is critical for their function. Regulatory role of an epigenetic reader lens epithelium-derived growth factor/p75 (LEDGF/p75) requires at least two copies of this protein to overcome the nucleosome-induced barrier to transcription elongation. Moreover, various LEDGF/p75 binding partners are enriched for dimeric features, further underscoring the functional regulatory role of LEDGF/p75 dimerization. Here, we dissected the minimal dimerization region in the C-terminal part of LEDGF/p75 and, using paramagnetic NMR spectroscopy, identified the key molecular contacts that helped to refine the solution structure of the dimer. The LEDGF/p75 dimeric assembly is stabilized by domain swapping within the integrase binding domain and additional electrostatic "stapling" of the negatively charged α helix formed in the intrinsically disordered C-terminal region. We validated the dimerization mechanism using structure-inspired dimerization defective LEDGF/p75 variants and chemical crosslinking coupled to mass spectrometry. We also show how dimerization might affect the LEDGF/p75 interactome. ispartof: STRUCTURE vol:28 issue:12 pages:1288-+ ispartof: location:United States status: published

Background Relapsed acute lymphoblastic leukemia (ALL) is one of the main causes of mortality in childhood malignancies. Previous genetic studies demonstrated that chemoresistant ALL is driven by activating mutations in NT5C2, the gene encoding cytosolic 5´-nucleotidase (cN-II). However, molecular mechanisms underlying this hyperactivation are still unknown. Here, we present kinetic and structural properties of cN-II variants that represent 75 % of mutated alleles in patients who experience relapsed ALL (R367Q, R238W and L375F). Results Enzyme kinetics measurements revealed that the mutants are consitutively active without need for allosteric activators. This shows that hyperactivity is not caused by a direct catalytic effect but rather by misregulation of cN-II. X-ray crystallography combined with mass spectrometry-based techniques demonstrated that this misregulation is driven by structural modulation of the oligomeric interface within the cN-II homotetrameric assembly. These specific conformational changes are shared between the studied variants, despite the relatively random spatial distribution of the mutations. Conclusions These findings define a common molecular mechanism for cN-II hyperactivity, which provides a solid basis for targeted therapy of leukemia. Our study highlights the cN-II oligomerization interface as an attractive pharmacological target. Electronic supplementary material The online version of this article (doi:10.1186/s12915-016-0313-y) contains supplementary material, which is available to authorized users.

Lens epithelium-derived growth factor (LEDGF/p75) is an epigenetic reader and attractive therapeutic target involved in HIV integration and the development of mixed lineage leukaemia (MLL1) fusion-driven leukaemia. Besides HIV integrase and the MLL1-menin complex, LEDGF/p75 interacts with various cellular proteins via its integrase binding domain (IBD). Here we present structural characterization of IBD interactions with transcriptional repressor JPO2 and domesticated transposase PogZ, and show that the PogZ interaction is nearly identical to the interaction of LEDGF/p75 with MLL1. The interaction with the IBD is maintained by an intrinsically disordered IBD-binding motif (IBM) common to all known cellular partners of LEDGF/p75. In addition, based on IBM conservation, we identify and validate IWS1 as a novel LEDGF/p75 interaction partner. Our results also reveal how HIV integrase efficiently displaces cellular binding partners from LEDGF/p75. Finally, the similar binding modes of LEDGF/p75 interaction partners represent a new challenge for the development of selective interaction inhibitors. ispartof: Nature Communications vol:6 pages:7968- ispartof: location:England status: published

Summary the active site, we have examined the effects of anti- HIV-1 Pr monoclonal antibodies (mAbs) on the catalytic Background: Since the demonstration that the prote- activityoftheprotease[9,10].Wehaverecentlyreportedase of the human immunodeficiency virus (HIV Pr) is a preliminary study of mAb1696, which, although raisedessential in the viral life cycle, this enzyme has become againsttheHIV-1Pr,inhibitsthecatalyticactivityofbothone of the primary targets for antiviral drug design. The the HIV-1 and HIV-2 enzymes with inhibition constantsmurinemonoclonalantibody1696(mAb1696),produced of 0.6 nM and 1.5 nM, respectively, at pH 7.4 [11]. Thisby immunization with the HIV-1 protease, inhibits the studyalsoshowedthatmAb1696cross-reactswithpep-catalytic activity of the enzyme of both the HIV-1 and tides containing the N terminus of the HIV protease. TheHIV-2 isolates with inhibition constants in the low nano- N-terminal region accounts for alarge percentage of themolar range. The antibody cross-reacts with peptides interface between the two HIV Pr monomers because itthat include the N terminus of the enzyme, a region that interdigitates with the C-terminal segment from theis highly conserved in sequence among different viral other monomer, thus forming a 4-stranded intermolecu-strains and that, furthermore, is crucial for homodimer- lar -pleated sheet in the active homodimer. We pro-ization to the active enzymatic form. posed that mAb1696 inhibits HIV Pr by perturbing thenative structure of the enzyme at the dimer interface

An E. coli plasmid expressing efficiently an artificial precursor of bovine leukemia virus (BLV) proteinase under transcriptional control of the phage T7 promoter was constructed. The expression product accumulates in the induced E. coli cells in the form of insoluble cytoplasmic inclusions. Solubilization of the inclusions and a refolding step yield almost pure and completely self-processed proteinase. Purification to homogeneity was achieved by ion-exchange chromatography and reverse-phase HPLC. On a preparative scale, a high yield of enzymatically active proteinase was obtained. An initial study using a series of synthetic peptide substrates shows a distinct substrate specificity of BLV proteinase.