Anita Changela

Crystal structure of a complex of a type IA DNA topoisomerase with a single-stranded DNA molecule.

A variety of cellular processes, including DNA replication, transcription, and chromosome condensation, require enzymes that can regulate the ensuing topological changes occurring in DNA. Such enzymes—DNA topoisomerases—alter DNA topology by catalysing the cleavage of single-stranded DNA (ssDNA) or double-stranded DNA (dsDNA), the passage of DNA through the resulting break, and the rejoining of the broken phosphodiester backbone. DNA topoisomerase III from Escherichia coli belongs to the type IA family of DNA topoisomerases, which transiently cleave ssDNA via formation of a covalent 5′ phosphotyrosine intermediate. Here we report the crystal structure, at 2.05 Å resolution, of an inactive mutant of E. coli DNA topoisomerase III in a non-covalent complex with an 8-base ssDNA molecule. The enzyme undergoes a conformational change that allows the oligonucleotide to bind within a groove leading to the active site. We note that the ssDNA molecule adopts a conformation like that of B-DNA while bound to the enzyme. The position of the DNA within the realigned active site provides insight into the role of several highly conserved residues during catalysis. These findings confirm various aspects of the type IA topoisomerase mechanism while suggesting functional implications for other topoisomerases and proteins that perform DNA rearrangements.

Structure and mechanism of the RNA triphosphatase component of mammalian mRNA capping enzyme

The 5′ capping of mammalian pre‐mRNAs is initiated by RNA triphosphatase, a member of the cysteine phosphatase superfamily. Here we report the 1.65 Å crystal structure of mouse RNA triphosphatase, which reveals a deep, positively charged active site pocket that can fit a 5′ triphosphate end. Structural, biochemical and mutational results show that despite sharing an HCxxxxxR(S/T) motif, a phosphoenzyme intermediate and a core α/β‐fold with other cysteine phosphatases, the mechanism of phosphoanhydride cleavage by mammalian capping enzyme differs from that used by protein phosphatases to hydrolyze phosphomonoesters. The most significant difference is the absence of a carboxylate general acid catalyst in RNA triphosphatase. Residues conserved uniquely among the RNA phosphatase subfamily are important for function in cap formation and are likely to play a role in substrate recognition.

Structure‐based design of robust glucose biosensors using a Thermotoga maritima periplasmic glucose‐binding protein

We report the design and engineering of a robust, reagentless fluorescent glucose biosensor based on the periplasmic glucose‐binding protein obtained from Thermotoga maritima (tmGBP). The gene for this protein was cloned from genomic DNA and overexpressed in Escherichia coli, the identity of its cognate sugar was confirmed, ligand binding was studied, and the structure of its glucose complex was solved to 1.7 Å resolution by X‐ray crystallography. TmGBP is specific for glucose and exhibits high thermostability (midpoint of thermal denaturation is 119 ± 1°C and 144 ± 2°C in the absence and presence of 1 mM glucose, respectively). A series of fluorescent conjugates was constructed by coupling single, environmentally sensitive fluorophores to unique cysteines introduced by site‐specific mutagenesis at positions predicted to be responsive to ligand‐induced conformational changes based on the structure. These conjugates were screened to identify engineered tmGBPs that function as reagentless fluorescent glucose biosensors. The Y13C•Cy5 conjugate is bright, gives a large response to glucose over concentration ranges appropriate for in vivo monitoring of blood glucose levels (1–30 mM), and can be immobilized in an orientation‐specific manner in microtiter plates to give a reversible response to glucose. The immobilized protein retains its response after long‐term storage at room temperature.

Structural Analysis of a Periplasmic Binding Protein in the Tripartite ATP-Independent Transporter Family Reveals a Tetrameric Assembly That May Have a Role in Ligand Transport

Several bacterial solute transport mechanisms involve members of the periplasmic binding protein (PBP) superfamily that bind and deliver ligand to integral membrane transport proteins in the ATP-binding cassette, tripartite tricarboxylate transporter, or tripartite ATP-independent (TRAP) families. PBPs involved in ATP-binding cassette transport systems have been well characterized, but only a few PBPs involved in TRAP transport have been studied. We have measured the thermal stability, determined the oligomerization state by small angle x-ray scattering, and solved the x-ray crystal structure to 1.9 Å resolution of a TRAP-PBP (open reading frame tm0322) from the hyperthermophilic bacterium Thermotoga maritima (TM0322). The overall fold of TM0322 is similar to other TRAP transport related PBPs, although the structural similarity of backbone atoms (2.5-3.1 Å root mean square deviation) is unusually low for PBPs within the same group. Individual monomers within the tetrameric asymmetric unit of TM0322 exhibit high root mean square deviation (0.9 Å) to each other as a consequence of conformational heterogeneity in their binding pockets. The gel filtration elution profile and the small angle x-ray scattering analysis indicate that TM0322 assembles as dimers in solution that in turn assemble into a dimer of dimers in the crystallographic asymmetric unit. Tetramerization has been previously observed in another TRAP-PBP (the Rhodobacter sphaeroides α-keto acid-binding protein) where quaternary structure formation is postulated to be an important requisite for the transmembrane transport process.

Protein-nucleotide interactions in E. coli DNA topoisomerase I.

DNA topoisomerases are the enzymes responsible for controlling and maintaining the topological states of DNA. Type IA enzymes work by transiently breaking the phosphodiester backbone of one strand to allow passage of another strand through the break. The protein has to perform complex rearrangements of the DNA, and hence it is likely that different regions of the enzyme bind DNA with different affinities. In order to identify some of the DNA binding sites in the protein, we have solved the structures of several complexes of the 67 kDa N-terminal fragment of Escherichia coli DNA topoisomerase I with mono- and trinucleotides. There are five different binding sites in the complexes, one of which is adjacent to the active site. Two other sites are in the central hole of the protein and may represent general DNA binding regions. The positions of these sites allow us to identify different DNA binding regions and to understand their possible roles in the catalytic cycle.

Structural Adaptations that Modulate Monosaccharide, Disaccharide, and Trisaccharide Specificities in Periplasmic Maltose-Binding Proteins

Periplasmic binding proteins comprise a superfamily that is present in archaea, prokaryotes, and eukaryotes. Periplasmic binding protein ligand-binding sites have diversified to bind a wide variety of ligands. Characterization of the structural mechanisms by which functional adaptation occurs is key to understanding the evolution of this important protein superfamily. Here we present the structure and ligand-binding properties of a maltotriose-binding protein identified from the Thermus thermophilus genome sequence. We found that this receptor has a high affinity for the trisaccharide maltotriose (K(d)<1 microM) but little affinity for disaccharides that are transported by a paralogous maltose transport operon present in T. thermophilus. Comparison of this structure to other proteins that adopt the maltose-binding protein fold but bind monosaccharides, disaccharides, or trisaccharides reveals the presence of four subsites that bind individual glucose ring units. Two loops and three helical segments encode adaptations that control the presence of each subsite by steric blocking or hydrogen bonding. We provide a model in which the energetics of long-range conformational equilibria controls subsite occupancy and ligand binding.

DNA manipulators: caught in the act

DNA is a dynamic molecule that undergoes constant changes in the cell through interactions with numerous proteins. Several classes of enzyme are specialized in promoting DNA rearrangements, including site-specific recombinases, DNA helicases, transposases and DNA topoisomerases. Recent structures of protein-DNA reaction intermediates trapped in various states of DNA remodeling, complemented by biochemical and biophysical functional studies, have enhanced our understanding of their respective mechanistic pathways.

Purification, crystallization and preliminary X-ray diffraction analysis of the human mismatch repair protein MutSβ.

MutSβ is a eukaryotic mismatch repair protein that preferentially targets extrahelical unpaired nucleotides and shares partial functional redundancy with MutSα (MSH2-MSH6). Although mismatch recognition by MutSα has been shown to involve a conserved Phe-X-Glu motif, little is known about the lesion-binding mechanism of MutSβ. Combined MSH3/MSH6 deficiency triggers a strong predisposition to cancer in mice and defects in msh2 and msh6 account for roughly half of hereditary nonpolyposis colorectal cancer mutations. These three MutS homologs are also believed to play a role in trinucleotide repeat instability, which is a hallmark of many neurodegenerative disorders. The baculovirus overexpression and purification of recombinant human MutSβ and three truncation mutants are presented here. Binding assays with heteroduplex DNA were carried out for biochemical characterization. Crystallization and preliminary X-ray diffraction analysis of the protein bound to a heteroduplex DNA substrate are also reported.