Structure of the extremely slow GTPase Rab6A in the GTP bound form at 1.8 Å resolution

Rab/Ypt GTPases represent a > 60 member large family of membrane traffic regulators in eukaryotic cells. Members of this group display intrinsic GTPase activity varying over two orders of magnitude. Here, we show that Rab6A represents the RabGTPase with the slowest spontaneous GTPase activity yet measured (5 × 10−6 s−1). Due to the very low intrinsic hydrolysis rate we were able to crystallise and solve the structure of the Rab6A:GTP complex to 1.82 Å resolution. Analysis of the structure suggests that low catalytic activity of the Rab6A might be due to high flexibility of the Switch II region and a low degree of constraint of critically important for catalysis Gln 72

Isotopic labeling of recombinant proteins expressed in the protozoan host Leishmania tarentolae

Abstract Isotope labeling of recombinant proteins is a prerequisite for application of nuclear magnetic resonance spectroscopy (NMR) for the characterization of the three-dimensional structures and dynamics of proteins. Overexpression of isotopically labeled proteins in bacterial or yeast host organisms has several drawbacks. In this work, we tested whether the recently described eukaryotic protein expression system based on the protozoa Leishmania tarentolae could be used for production of amino acid speciWc 15 N-labeled recombinant proteins. Using synthetic growth medium we were able to express in L. tarentolae and purify to homogeneity (15)N-valine labeled Enchanced Green Fluorescent Protein (EGFP) with the Wnal yield of 5.7 mg/liter of suspension culture. NMR study of isolated EGFP illustrated the success of the labeling procedure allowing identiWcation of all 18 valine residues of the protein in the HSQC spectrum. Our results demonstrate the suitability of the L. tarentolae expression system for production of isotopically labeled proteins. © 2006 Elsevier Inc. All rights reserved. Keywords: 15 N-labeling; Recombinant protein; Eukaryotic expression system Nuclear magnetic resonance spectroscopy (NMR) 1 is one of two existing methods that allow determination of protein structure at atomic resolution. A majority of NMR techniques in biology require isotopic labeling ( 2 H, 13 C, and/or 15 N) of recombinant proteins. Currently, most isotopically labeled recombinant proteins are expressed heterologously in Escherichia coli. Despite its obvious advantages such as rapid growth, developed methods of protein expression and cheapness of cultivation E. coli has a range of shortcomings that limits its utility in protein studies. The most prominent problem relates to ineYciency of E. coli to assist folding of eukaryotic polypeptides producing only ca. 15% of eukaryotic proteins in their active form We recently described a new protein expression system based on the non-pathogenic trypanosomatid Leishmani

The Interaction between Cyclin B1 and Cytomegalovirus Protein Kinase pUL97 is Determined by an Active Kinase Domain

Replication of human cytomegalovirus (HCMV) is characterized by a tight virus-host cell interaction. Cyclin-dependent protein kinases (CDKs) are functionally integrated into viral gene expression and protein modification. The HCMV-encoded protein kinase pUL97 acts as a CDK ortholog showing structural and functional similarities. Recently, we reported an interaction between pUL97 kinase with a subset of host cyclins, in particular with cyclin T1. Here, we describe an interaction of pUL97 at an even higher affinity with cyclin B1. As a striking feature, the interaction between pUL97 and cyclin B1 proved to be strictly dependent on pUL97 activity, as interaction could be abrogated by treatment with pUL97 inhibitors or by inserting mutations into the conserved kinase domain or the nonconserved C-terminus of pUL97, both producing loss of activity. Thus, we postulate that the mechanism of pUL97-cyclin B1 interaction is determined by an active pUL97 kinase domain

Biophysical analysis of the interaction of Rab6a GTPase with its effector domains

Rab GTPases are key regulators of intracellular vesicular transport that control vesicle budding, cargo sorting, transport, tethering, and fusion. In the inactive (GDP-bound) conformation, Rab GTPases are targeted to intracellular compartments where they are converted into the active GTP-bound form and recruit effector domain containing proteins. Rab6a has been implicated in dynein-mediated vesicle movement at the Golgi apparatus and shown to interact with a plethora of effector proteins. In this study, we identify minimal Rab6a binding domains of three Rab6a effector proteins: PIST, BicaudalD2, and p150. All three domains are >15-kDa fragments predicted to form coiled-coil structures that display no sequence homology to each other. Complex formation with BicaudalD2 and p150 has a moderate inhibitory effect on the intrinsic GTPase activity of Rab6a, while interaction with PIST has no influence on the hydrolysis rate. The effectors bind activated Rab6a with comparable affinities with K values ranging from high nanomolar to low micromolar. Transient kinetic analysis revealed that effectors bind to Rab6a in an apparent single-step reaction characterized by relatively rapid on- and off-rates. We propose that the high off-rates of Rab6·effector complexes enable GTPase-activating protein-mediated net dissociation, which would not be possible if the off-rate were significantly slower

OCT4 interprets and enhances nucleosome flexibility.

Funder: Royal Netherlands Academy of Arts and SciencesFunder: Max Planck SocietyFunder: University of UtrechtFunder: Babes-Bolyai UniversityFunder: Gauss Centre for Supercomputing e.V.Funder: Max Planck Computing and Data FacilityPioneer transcription factors are proteins that induce cellular identity transitions by binding to inaccessible regions of DNA in nuclear chromatin. They contribute to chromatin opening and recruit other factors to regulatory DNA elements. The structural features and dynamics modulating their interaction with nucleosomes are still unresolved. From a combination of experiments and molecular simulations, we reveal here how the pioneer factor and master regulator of pluripotency, Oct4, interprets and enhances nucleosome structural flexibility. The magnitude of Oct4's impact on nucleosome dynamics depends on the binding site position and the mobility of the unstructured tails of nucleosomal histone proteins. Oct4 uses both its DNA binding domains to propagate and stabilize open nucleosome conformations, one for specific sequence recognition and the other for nonspecific interactions with nearby regions of DNA. Our findings provide a structural basis for the versatility of transcription factors in engaging with nucleosomes and have implications for understanding how pioneer factors induce chromatin dynamics