The American Crystallographic Association (ACA) has elected Bruce Foxman, Professor of Chemistry Emeritus, as a member of the 2020 class of ACA Fellows. The ACA recognized his leadership of the solid-state chemistry research community and his research in topotactic relationships, solid-state dimerization and polymerization, and polymorphism. Foxman has also contributed extensively to the development of new methods for X-ray crystal structure determination. Bruce joined the Brandeis faculty in 1972 and is still research-active and also collaborating with many colleagues at Brandeis and elsewhere. He is a superb teacher (2009 Brandeis Prize for Excellence in Teaching) and has developed widely-used downloadable tutorials on Symmetry and Space Groups and An Introduction to X-ray Structure Determination for High School Students.
Bruce Foxman Elected American Crystallographic Association Fellow
ACA Symposium to honor Foxman
At the 2012 Meeting of the American Crystallographic Association, to be held in Boston starting this weekend, one of the highlights will be a session entitled “Transactions: Transformations and Structural Oddities in Molecular Crystals: In Honor of Bruce M. Foxman“. This session, organized to honor \Professor of Chemistry Bruce Foxman “for his contributions to the field of solid state chemistry and his dedication to teaching” on the occasion of his 7oth birthday. Foxman’s research over the years has involved solid state reactions and polymorphism of molecular crystals, and one of his greatest contributions to the field is a series of online tutorials, including one on Symmetry and Space Groups, another on Bruker’s APEX 2 software. and a third aimed at high school students. The symposium will be held in two parts on Sunday July 29, and Wednesday, August 1, at the ACA meeting in Boston.
Protein Flexing: A New Look at Transcription-Coupled DNA Repair
Alexandra M. Deaconescu, a research associate in the Rosenstiel Basic Medical Sciences Research Center and 2008-2010 Fellow of the Damon Runyon Cancer Research Foundation, together with Professor of Biochemistry and HHMI Investigator Nikolaus Grigorieff and collaborators in the Laboratory of Dr. Irina Artsimovitch at Ohio State University have just published a new study in PNAS, which delineates novel mechanistic details of transcription-coupled DNA repair.
In any cell, there is intense interplay between various DNA-based transactions, such as replication, transcription and DNA repair. More than twenty years ago, it was discovered that DNA lesions that cause stalling of RNA polymerase molecules elicit a form of preferential nucleotide excision repair (NER) that exists in both eubacteria and eukaryotes, and specifically targets the transcribed DNA strand. Termed transcription-coupled DNA repair (TCR), the process is found to be carried out in bacteria by an ATPase called Mfd or TRCF (see Figure, right). In TCR, TRCF performs two functions: 1) it recognizes a damage-stalled RNA polymerase (RNAP), then dissociates it off the DNA using energy derived from ATP hydrolysis and 2) it recruits DNA repair enzymes via binding to the UvrA subunit of the Uvr(A)BC NER machinery [1]. The Uvr(A)BC machinery is one of the main players in bacterial DNA repair, and distinguishes itself from other DNA repair proteins by its ability to repair a remarkably diverse repertoire of lesions by utilizing a “cut and patch” mechanism, whereby an oligonucleotide containing the damage is excised and the gap later filled.
The cellular role of TRCFs extends beyond TCR. Because of their ability to forward translocate and dissociate stalled RNAPs (or “backtracked” RNAPs that have slid backwards on the template) [2], TRCFs are also involved in transcription elongation regulation [3, 4], resolution of head-on collisions of the transcription apparatus with the DNA replication machinery [5], and antibiotic resistance [6, 7]. In humans, the effects of impaired TCR are systemic and complex. Mutations in the transcription-repair coupling factor CSB lead to Cockayne Syndrome [8], a progeroid (accelerated-aging) disease characterized by severe developmental abnormalities and neurodegeneration, and whose etiology is currently poorly understood.
To elucidate the mechanism underpinning UvrA recruitment by TRCF, Deaconescu crystallized and solved the X-ray structure of a core UvrA-TRCF complex (Figure, left) demonstrating that UvrA binding involves unmasking of a conserved intramolecular surface within TRCF via a gating motion of the C-terminal domain (red in Figure above). Despite significant effort so far, Deaconescu is still trying to coax nucleotide-bound TRCF to form crystals suitable for X-ray diffraction. These would be highly informative because ATP is required for DNA binding, and its hydrolysis leads to TRCF translocation on dsDNA and ultimately release of RNAP off the 
damaged template. Because diffracting crystals eluded her, and to further find out how ADP/ATP modulate the structure of TRCF, Deaconescu learned small-angle X-ray scattering techniques suitable for probing TRCF in solution in the absence and presence of nucleotides, thus circumventing the need for highly-ordered crystals. Then, the Brandeis team and their collaborators at Ohio State employed domain-locking disulfide engineering in conjunction with functional assays to gain a deeper understanding of what TRCF looks like during its catalytic cycle and upon binding to UvrA. They find that the two main functions of TRCF (RNAP release and UvrA binding) can be uncoupled, suggesting that UvrA recruitment may only occur during/post RNAP release, and not upon RNAP binding as had been proposed earlier in the literature [9]. Furthermore, they show that the ternary elongation complex (consisting of RNAP, template and nascent RNA), but not naked DNA, significantly stimulates ATP hydrolysis by TRCF. Thus, bacterial TRCF operates in a manner reminiscent of that utilized by eukaryotic chromatin remodeling factors, and are preferentially stimulated by nucleosomes over naked DNA substrates.
Deaconescu previously “looked” at TCR using X-rays – as a graduate student she solved the first structure of an intact transcription-repair coupling factor from any organism using X-ray crystallography [10]. She now hopes to reconstitute the larger intermediates that form during TCR and bridge low- with high-resolution information using hybrid structural methods, particularly electron cryo-microscopy, and ultimately formulate a cogent model of how TRCFs operate in cells.
Mapping hydrogens in chymotrypsin structures with neutron diffraction
In a new paper “Time-of-flight neutron diffraction study of bovine γ-chymotrypsin at the Protein Crystallography Station” published in this month’s edition of the journal Acta Cryst F, Biochemistry grad student Louis Lazar and co-workers from the Petsko-Ringe lab report progress on their project to determine exact hydrogen positions in proteins using neutron diffraction.
Neutron diffraction was chosen, as opposed to X-ray diffraction, because one can visualize hydrogen species directly using neutrons, while it is extremely difficult and in most cases impossible to do so using X-ray diffraction. They chose the protein γ-chymotrypsin in order to determine hydrogen positions, as it fills the necessary requirements to be suitable for a neutron diffraction experiment. These requirements include a very large crystal size (> 1 mm3), moderately sized unit cell axes (no dimension greater than 100 Å), and it must be very stable as well as well-characterized. γ-chymotrypsin is the stereotypical serine protease, cleaving C-terminal to aliphatic and aromatic residues and containing a catalytic triad of serine, histidine, and aspartate. This information on hydrogen placement can then be applied to improve computational methods in which said placement is paramount, such as molecular modeling and rational drug design.
The paper details the collection of neutron data at pD (pH*) 7.1, with the help of the scientists at the Los Alamos National Laboratory. In particular, from the initial maps, they note that the catalytic histidine is doubly protonated, while the serine and aspartate making up the catalytic triad do not show density for the presence of deuterium. In order to complete the study of γ-chymotrypsin, data at a variety of pH values must be collected; data at pD (pH*) 5.6 has already been collected (Acta Cryst F65, 317-320), and data at pD (pH*) 9.0 will be collected in the future.
see also: full text of article (Brandeis users)
