ABSTRACT
Modification of cellular proteins by the ubiquitin-like protein SUMO is essential for nuclear processes and cell cycle progression in yeast. The Ulp1 protease catalyzes two essential functions in the SUMO pathway: (1) processing of full-length SUMO to its mature form and (2) deconjugation of SUMO from targeted proteins. Selective reduction of the proteolytic reaction produced a covalent thiohemiacetal transition state complex between a Ulp1 C-terminal fragment and its cellular substrate Smt3, the yeast SUMO homolog. The Ulp1-Smt3 crystal structure and functional testing of elements within the conserved interface elucidate determinants of SUMO recognition, processing, and deconjugation. Genetic analysis guided by the structure further reveals a regulatory element N-terminal to the proteolytic domain that is required for cell growth in yeast.
Subject(s)
Cysteine Endopeptidases/chemistry , Fungal Proteins/chemistry , Small Ubiquitin-Related Modifier Proteins , Ubiquitins/chemistry , Amino Acid Sequence , Catalytic Domain , Crystallography, X-Ray , Cysteine Endopeptidases/metabolism , Glycine , Models, Molecular , Molecular Sequence Data , Peptide Fragments/chemistry , Saccharomyces cerevisiae , Sequence Homology, Amino Acid , Substrate Specificity , Surface PropertiesABSTRACT
ParE is the ATP-binding subunit of topoisomerase IV (Topo IV). During topoisomerization, the ATP-binding and hydrolysis cycle must be coordinated with the cycle of DNA cleavage and religation. We have isolated three dominant-negative mutant alleles of parE that encode ParE proteins that fail to hydrolyze ATP when reconstituted with ParC to form Topo IV. ParE G110S Topo IV and ParE S123L Topo IV failed to bind ATP at all, whereas ParE T201A could bind ATP. All three mutant Topo IV proteins exhibited an elevated level of spontaneous DNA cleavage that could be associated with a decreased rate of DNA resealing. In ParE T201A Topo IV, this defect appeared to result from an increased likelihood that the tetrameric enzyme would fall apart after DNA cleavage. Thus, while ATP is not required for DNA cleavage, the properties of these mutant enzymes suggests that ATP-hydrolysis informs DNA religation.
Subject(s)
Adenosine Triphosphatases/genetics , Bacterial Proteins/genetics , DNA Topoisomerases, Type II/genetics , DNA-Binding Proteins/genetics , Escherichia coli/enzymology , Adenosine Triphosphate/metabolism , Adenylyl Imidodiphosphate/metabolism , Animals , Chromatography, Gel , Crithidia fasciculata , DNA Mutational Analysis , DNA Topoisomerase IV , DNA, Superhelical/metabolism , Dimerization , Models, Molecular , MutationABSTRACT
In order to define regions of ParE, one of the two subunits of topoisomerase IV, that are involved in catalysis during topoisomerization, we developed a selection procedure to isolate dominant-negative parE alleles. Both wild-type parC and mutagenized parE were expressed from a tightly-regulated lac promoter on a moderate-copy plasmid. Mutated parE alleles were rescued from those plasmids that caused IPTG-dependent cell death. The mutant ParE proteins could be divided into two groups when reconstituted with ParC to form topoisomerase IV, those that elicited hyper-DNA cleavage and those that affected covalent complex formation.
Subject(s)
Bacterial Proteins/genetics , DNA Topoisomerases, Type II/genetics , DNA-Binding Proteins/genetics , Escherichia coli/enzymology , Alleles , DNA Topoisomerase IV , DNA, Superhelical/chemistry , Electrophoresis, Polyacrylamide Gel , Isopropyl Thiogalactoside/metabolism , Microscopy, Fluorescence , Mutation , PhenotypeABSTRACT
The products of three dominant-negative alleles of parE, encoding the ATP-binding subunit of topoisomerase IV (Topo IV), were purified and their activities characterized when reconstituted with ParC to form Topo IV. The ability of the ParE E418K, ParE G419D, and ParE G442D mutant Topo IVs to bind DNA, hydrolyze ATP, and close their ATP-dependent clamp was relatively unaffected. However, their ability to relax negatively supercoiled DNA was compromised significantly. This could be attributed to severe defects in covalent complex formation between ParC and DNA. Thus, these residues, which are far from the active site Tyr of ParC, contribute to covalent catalysis. This indicates that a dramatic conformational rearrangement of the protein likely occurs subsequent to the binding of the G segment at the DNA gate and prior to its opening.
Subject(s)
Bacterial Proteins/genetics , DNA Topoisomerases, Type II/genetics , DNA-Binding Proteins/genetics , Escherichia coli/enzymology , Adenosine Triphosphate/metabolism , Alleles , Bacterial Proteins/chemistry , Binding Sites , DNA Topoisomerase IV , DNA Topoisomerases, Type II/chemistry , DNA, Superhelical/metabolism , DNA-Binding Proteins/chemistry , Models, Molecular , Mutation , Norfloxacin/chemistry , Protein Binding/genetics , Protein ConformationABSTRACT
Sec7-related guanine nucleotide exchange factors (GEFs) initiate vesicle budding from the Golgi membrane surface by converting the GTPase ARF to a GTP-bound, membrane-associated form. Here we report the crystal structure of the catalytic Sec7 homology domain of Arno, a human GEF for ARF1, determined at 2.2 angstroms resolution. The Sec7 domain is an elongated, all-helical protein with a distinctive hydrophobic groove that is phylogenetically conserved. Structure-based mutagenesis identifies the groove and an adjacent conserved loop as the ARF-interacting surface. The sites of Sec7 domain interaction on ARF1 have subsequently been mapped, by protein footprinting experiments, to the switch 1 and switch 2 GTPase regions, leading to a model for the interaction between ARF GTPases and Sec7 domain exchange factors.
