David T. Gallagher

The crystal structure of chorismate lyase shows a new fold and a tightly retained product.

The enzyme chorismate lyase (CL) catalyzes the removal of pyruvate from chorismate to produce 4‐hydroxy benzoate (4HB) for the ubiquinone pathway. In Escherichia coli, CL is monomeric, with 164 residues. We have determined the structure of the CL product complex by crystallographic heavy‐atom methods and report the structure at 1.4‐Å resolution for a fully active double Cys‐to‐Ser mutant and at 2.0‐Å resolution for the wild‐type. The fold involves a 6‐stranded antiparallel β‐sheet with no spanning helices and novel connectivity. The product is bound internally, adjacent to the sheet, with its polar groups coordinated by two main‐chain amides and by the buried side‐chains of Arg 76 and Glu 155. The 4HB is completely sequestered from solvent in a largely hydrophobic environment behind two helix–turn–helix loops. The extensive product binding that is observed is consistent with biochemical measurements of slow product release and 10‐fold stronger binding of product than substrate. Substrate binding and kinetically rate‐limiting product release apparently require the rearrangement of these active‐site‐covering loops. Implications for the biological function of the high product binding are considered in light of the unique cellular role of 4HB, which is produced by cytoplasmic CL but is used by the membrane‐bound enzyme 4HB octaprenyltransferase. Proteins 2001;44:304–311.

Chorismate lyase: kinetics and engineering for stability.

By removing the enolpyruvyl group from chorismate, chorismate lyase (CL) produces p-hydroxybenzoate (p-HB) for the ubiquinone biosynthetic pathway. We have analyzed CL by several spectroscopic and chemical techniques and measured its kinetic (kcat=1.7 s(-1), K(m)=29 microM) and product inhibition parameters (K(p)=2.1 microM for p-HB). Protein aggregation, a serious problem with wild type CL, proved to be primarily due to the presence of two surface-active cysteines, whose chemical modification or mutation (to serines) gave greatly improved solution behavior and minor effects on enzyme activity. CL is strongly inhibited by its product p-HB; for this reason activity and inhibition measurements were analyzed by both initial rate and progress curve methods. The results are consistent, but in this case where the stable enzyme-product complex rapidly becomes the predominant form of the enzyme, progress curve methods are more efficient. We also report inhibition measurements with several substrate and product analogs that give information on ligand binding interactions of the active site. The biological function of the unusual product retention remains uncertain, but may involve a mechanism of directed delivery to the membrane-bound enzyme that follows CL in the ubiquinone pathway.

The Biomolecular Crystallization Database Version 4: expanded content and new features.

The Biological Macromolecular Crystallization Database (BMCD) has been a publicly available resource since 1988, providing a curated archive of information on crystal growth for proteins and other biological macromolecules. The BMCD content has recently been expanded to include 14 372 crystal entries. The resource continues to be freely available at http://xpdb.nist.gov:8060/BMCD4. In addition, the software has been adapted to support the Java-based Lucene query language, enabling detailed searching over specific parameters, and explicit search of parameter ranges is offered for five numeric variables. Extensive tools have been developed for import and handling of data from the RCSB Protein Data Bank. The updated BMCD is called version 4.02 or BMCD4. BMCD4 entries have been expanded to include macromolecule sequence, enabling more elaborate analysis of relations among protein properties, crystal-growth conditions and the geometric and diffraction properties of the crystals. The BMCD version 4.02 contains greatly expanded content and enhanced search capabilities to facilitate scientific analysis and design of crystal-growth strategies.

Profound Asymmetry in the Structure of the cAMP-free cAMP Receptor Protein (CRP) from Mycobacterium tuberculosis

The cyclic AMP receptor protein (CRP, also called catabolite gene activator protein or CAP) plays a key role in metabolic regulation in bacteria and has become a widely studied model allosteric transcription factor. On binding its effector cAMP in the N-terminal domain, CRP undergoes a structural transition to a conformation capable of specific DNA binding in the C-terminal domain and transcription initiation. The crystal structures of Escherichia coli CRP (EcCRP) in the cAMP-bound state, both with and without DNA, are known, although its structure in the off state (cAMP-free, apoCRP) remains unknown. We describe the crystal structure at 2.0Å resolution of the cAMP-free CRP homodimer from Mycobacterium tuberculosis H37Rv (MtbCRP), whose sequence is 30% identical with EcCRP, as the first reported structure of an off-state CRP. The overall structure is similar to that seen for the cAMP-bound EcCRP, but the apo MtbCRP homodimer displays a unique level of asymmetry, with a root mean square deviation of 3.5Å between all Cα positions in the two subunits. Unlike structures of on-state EcCRP and other homologs in which the C-domains are asymmetrically positioned but possess the same internal conformation, the two C-domains of apo MtbCRP differ both in hinge structure and in internal arrangement, with numerous residues that have completely different local environments and hydrogen bond interactions, especially in the hinge and DNA-binding regions. Comparison of the structures of apo MtbCRP and DNA-bound EcCRP shows how DNA binding would be inhibited in the absence of cAMP and supports a mechanism involving functional asymmetry in apoCRP.

Crystallization and phasing of alanine dehydrogenase from Archaeoglobus fulgidus

Alanine dehydrogenase (AlaDH) from the hyperthermophilic archaeon Archaeoglobus fulgidus is a dimer of 35 kDa chains. The archaeal enzyme appears to represent a new class of AlaDH that is not homologous to bacterial AlaDH enzymes, but has close evolutionary links to the broad ornithine cyclodeaminase/micro-crystallin family, which includes human thyroid hormone binding protein, which has 30% sequence identity to the A. fulgidus gene. The enzyme has been cloned, shown to catalyze the NAD-dependent interconversion of alanine and pyruvate and crystallized in several forms. Although the purified protein crystallized readily under many conditions, most of the crystals diffracted weakly or not at all. One polymorph growing in space group P2(1)2(1)2(1) has non-crystallographic symmetry that becomes crystallographic, changing the space group to P2(1)2(1)2, upon binding iridium or samarium. Before and after derivatization, these crystals diffracted to 2.5 A using synchrotron radiation. Multiwavelength diffraction data were collected from the non-isomorphous iridium derivative, enabling structure determination.

Structure of C73G putidaredoxin from Pseudomonas putida

The structure of the C73G mutant of putidaredoxin (Pdx), the Fe(2)S(2) ferredoxin that supplies electrons to cytochrome CYP101 (p450cam) for camphor oxidation, is reported at 1.9 A resolution in a C2 crystal form. The structure was solved by single-wavelength iron anomalous diffraction, which yielded electron density above the 2sigma level for over 97% of the non-H atoms in the protein. The final structure with R = 0.19 and R(free) = 0.21 has been deposited in the Protein Data Bank with accession code 1r7s. The C2 crystal contains three Pdx molecules in the asymmetric unit, giving three independent models of the protein that are very similar (r.m.s.d. < 0.3 A for the 106 C(alpha) atoms). The unusually high solvent fraction of 80% results in comparatively few crystal-packing artifacts. The structure is briefly compared with the recently reported crystal structures of the C73S and C73S/C85S mutants. In general, the eight independent molecules in the three crystal structures (three in C73G, three in C73S and two in C73S/C85S) are much more similar to each other than to the previously reported NMR structure of wild-type Pdx in solution. The present findings show a unanimous structure in some regions crucial for electron-transfer interactions, including the cluster-binding loop 39-48 and the cytochrome-interaction region of Asp38 and Trp106. In addition, the Cys45 amide group donates a hydrogen bond to cluster sulfur S1, with Ala46 adopting an Lalpha conformation, in all three molecules in the crystal.

Synchrotron white-beam X-ray topography of ribonuclease S crystals

With careful experimental design, indexed synchrotron white-beam X-ray topographs of ribonuclease S crystals at ambient temperature could be recorded with a definition and contrast comparable to that of monochromatic beam topographs of other proteins reported in the literature. By excluding wavelengths longer than 1 A from the white beam with a filter, a radiation dose equivalent to that required to record about 18 topographs could be tolerated without appreciable radiation damage to the samples. Bragg angles of 0.5 degrees or less were required to select low-index harmonically pure reflections with high intensities and extinction lengths only several times the samples thickness. The resulting X-ray topographs in some cases showed topographic detail and in others showed the even featureless background that has been considered characteristic of a protein crystal of low mosaicity. The ribonuclease S crystals were well ordered single crystals of a quality comparable to other protein crystals that have been studied by X-ray topography.

Polymorphous crystallization and diffraction of threonine deaminase from Escherichia coli

The biosynthetic threonine deaminase from Escherichia coli, an allosteric tetramer with key regulatory functions, has been crystallized in several crystal forms. Two distinct forms, both belonging to either space group P3121 or P3221, with different sized asymmetric units that both contain a tetramer, grow under identical conditions. Diffraction data sets to 2.8 A resolution (native) and 2. 9 A resolution (isomorphous uranyl derivative) have been collected from a third crystal form in space group I222.

Data on crystal organization in the structure of the Fab fragment from the NIST reference antibody, RM 8671

The reported data describe the crystallization, crystal packing, structure determination and twinning of the unliganded Fab (antigen-binding fragment) from the NISTmAb (standard reference material 8671). The raw atomic coordinates are available as Protein Data Bank structure 5K8A and biological aspects are described in the article, (Karageorgos et al., 2017) [1]. Crystal data show that the packing is unique, and show the basis for the crystals twinned growth. Twinning is a common and often serious problem in protein structure determination by x-ray crystallography [2]. In the present case the twinning is due to a small deviation (about 0.3 nm) from 4-fold symmetry in the primary intermolecular interface. The deviation produces pseudosymmetry, generating slightly different conformations of the protein, and alternating strong and weak forms of key packing interfaces throughout the lattice.