Mariusz Jaskolski

Crystal structure of a monomeric retroviral protease solved by protein folding game players

Following the failure of a wide range of attempts to solve the crystal structure of M-PMV retroviral protease by molecular replacement, we challenged players of the protein folding game Foldit to produce accurate models of the protein. Remarkably, Foldit players were able to generate models of sufficient quality for successful molecular replacement and subsequent structure determination. The refined structure provides new insights for the design of antiretroviral drugs.

The catalytic domain of avian sarcoma virus integrase: conformation of the active-site residues in the presence of divalent cations

BACKGROUND Members of the structurally-related superfamily of enzymes that includes RNase H, RuvC resolvase, MuA transposase, and retroviral integrase require divalent cations for enzymatic activity. So far, cation positions are reported in the X-ray crystal structures of only two of these proteins, E. coli and human immunodeficiency virus 1 (HIV-1) RNase H. Details of the placement of metal ions in the active site of retroviral integrases are necessary for the understanding of the catalytic mechanism of these enzymes. RESULTS The structure of the enzymatically active catalytic domain (residues 52-207) of avian sarcoma virus integrase (ASV IN) has been solved in the presence of divalent cations (Mn2+ or Mg2+), at 1.7-2.2 A resolution. A single ion of either type interacts with the carboxylate groups of the active site aspartates and uses four water molecules to complete its octahedral coordination. The placement of the aspartate side chains and metal ions is very similar to that observed in the RNase H members of this superfamily; however, the conformation of the catalytic aspartates in the active site of ASV IN differs significantly from that reported for the analogous residues in HIV-1 IN. CONCLUSIONS Binding of the required metal ions does not lead to significant structural modifications in the active site of the catalytic domain of ASV IN. This indicates that at least one metal-binding site is preformed in the structure, and suggests that the observed constellation of the acidic residues represents a catalytically competent active site. Only a single divalent cation was observed even at extremely high concentrations of the metals. We conclude that either only one metal ion is needed for catalysis, or that a second metal-binding site can only exist in the presence of substrate and/or other domains of the protein. The unexpected differences between the active sites of ASV IN and HIV-1 IN remain unexplained; they may reflect the effects of crystal contacts on the active site of HIV-1 IN, or a tendency for structural polymorphism.

Protein crystallography for non-crystallographers, or how to get the best (but not more) from published macromolecular structures

The number of macromolecular structures deposited in the Protein Data Bank now exceeds 45 000, with the vast majority determined using crystallographic methods. Thousands of studies describing such structures have been published in the scientific literature, and 14 Nobel prizes in chemistry or medicine have been awarded to protein crystallographers. As important as these structures are for understanding the processes that take place in living organisms and also for practical applications such as drug design, many non‐crystallographers still have problems with critical evaluation of the structural literature data. This review attempts to provide a brief outline of technical aspects of crystallography and to explain the meaning of some parameters that should be evaluated by users of macromolecular structures in order to interpret, but not over‐interpret, the information present in the coordinate files and in their description. A discussion of the extent of the information that can be gleaned from the coordinates of structures solved at different resolution, as well as problems and pitfalls encountered in structure determination and interpretation are also covered.

EARLY FLOWERING4 Recruitment of EARLY FLOWERING3 in the Nucleus Sustains the Arabidopsis Circadian Clock

ELF3 and ELF4 play pivotal roles in the circadian clock mechanism and in the integration of light signals to the clock, but the molecular basis of ELF3 and ELF4 action is poorly understood. This work uses multidisciplinary approaches to identify and characterize these clock factors as members of a dusk complex that works as a repressor to sustain rhythms of the circadian oscillator. The plant circadian clock is proposed to be a network of several interconnected feedback loops, and loss of any component leads to changes in oscillator speed. We previously reported that Arabidopsis thaliana EARLY FLOWERING4 (ELF4) is required to sustain this oscillator and that the elf4 mutant is arrhythmic. This phenotype is shared with both elf3 and lux. Here, we show that overexpression of either ELF3 or LUX ARRHYTHMO (LUX) complements the elf4 mutant phenotype. Furthermore, ELF4 causes ELF3 to form foci in the nucleus. We used expression data to direct a mathematical position of ELF3 in the clock network. This revealed direct effects on the morning clock gene PRR9, and we determined association of ELF3 to a conserved region of the PRR9 promoter. A cis-element in this region was suggestive of ELF3 recruitment by the transcription factor LUX, consistent with both ELF3 and LUX acting genetically downstream of ELF4. Taken together, using integrated approaches, we identified ELF4/ELF3 together with LUX to be pivotal for sustenance of plant circadian rhythms.

Crystal engineering with hydrogen bonds and halogen bonds

Molecular tapes mediated via strong O–H⋯N hydrogen bonds and weak C–I⋯O interactions are present in complexes of 4-nitrobenzoic acid·4-iodopyridine and 3,5-dinitrobenzoic acid·4-iodopyridine. Crystal structure of 4-nitrobenzamide·4-iodobenzamide has amide dimer tape and iodo⋯nitro interaction in orthogonal directions. There is good structural insulation in the hydrogen bonding and halogen bonding domains in these crystal structures. However, the less polarizable bromine and chlorine atoms show cross-pairing interactions that are less predictable. These preliminary results show that both carboxylic acid⋯pyridine and iodo⋯nitro heterosynthons may be simultaneously exploited for crystal design.

Fibrillogenic Oligomers of Human Cystatin C Are Formed by Propagated Domain Swapping

Cystatin C and the prion protein have been shown to form dimers via three-dimensional domain swapping, and this process has also been hypothesized to be involved in amyloidogenesis. Production of oligomers of other amyloidogenic proteins has been reported to precede fibril formation, suggesting oligomers as intermediates in fibrillogenesis. A variant of cystatin C, with a Leu68→ Gln substitution, is highly amyloidogenic, and carriers of this mutation suffer from massive cerebral amyloidosis leading to brain hemorrhage and death in early adulthood. This work describes doughnut-shaped oligomers formed by wild type and L68Q cystatin C upon incubation of the monomeric proteins. Purified oligomers of cystatin C are shown to fibrillize faster and at a lower concentration than the monomeric protein, indicating a role of the oligomers as fibril-assembly intermediates. Moreover, the present work demonstrates that three-dimensional domain swapping is involved in the formation of the oligomers, because variants of monomeric cystatin C, stabilized against three-dimensional domain swapping by engineered disulfide bonds, do not produce oligomers upon incubation under non-reducing conditions. Redox experiments using wild type and stabilized cystatin C strongly suggest that the oligomers, and thus probably the fibrils as well, are formed by propagated domain swapping rather than by assembly of domain-swapped cystatin C dimers.

Structural and functional aspects of PR-10 proteins

Physical, chemical and biological stress factors, such as microbial infection, upregulate the transcription levels of a number of plant genes, coding for the so‐called pathogenesis‐related (PR) proteins. For PR proteins of class‐10 (PR‐10), the biological function remains unclear, despite two decades of scientific research. PR‐10 proteins have a wide distribution throughout the plant kingdom and the class members share size and secondary structure organization. Throughout the years, we and other groups have determined the structures of a number of PR‐10 proteins, both in the crystalline state by X‐ray diffraction and in solution by NMR spectroscopy. Despite the accumulating structural information, our understanding of PR‐10 function is still limited. PR‐10 proteins are rather small (~ 160 amino acids) with a fold consisting of three α helices and seven antiparallel β strands. These structural elements enclose a large hydrophobic cavity that is most probably the key to their functional relevance. Also, the outer surface of these proteins is of extreme interest, as epitopes from a PR‐10 subclass cause allergic reactions in humans.

Crystal Structure of Vigna radiata Cytokinin-Specific Binding Protein in Complex with Zeatin

The cytosolic fraction of Vigna radiata contains a 17-kD protein that binds plant hormones from the cytokinin group, such as zeatin. Using recombinant protein and isothermal titration calorimetry as well as fluorescence measurements coupled with ligand displacement, we have reexamined the Kd values and show them to range from ∼10−6 M (for 4PU30) to 10−4 M (for zeatin) for 1:1 stoichiometry complexes. In addition, we have crystallized this cytokinin-specific binding protein (Vr CSBP) in complex with zeatin and refined the structure to 1.2 Å resolution. Structurally, Vr CSBP is similar to plant pathogenesis-related class 10 (PR-10) proteins, despite low sequence identity (<20%). This unusual fold conservation reinforces the notion that classic PR-10 proteins have evolved to bind small-molecule ligands. The fold consists of an antiparallel β-sheet wrapped around a C-terminal α-helix, with two short α-helices closing a cavity formed within the protein core. In each of the four independent CSBP molecules, there is a zeatin ligand located deep in the cavity with conserved conformation and protein–ligand interactions. In three cases, an additional zeatin molecule is found in variable orientation but with excellent definition in electron density, which plugs the entrance to the binding pocket, sealing the inner molecule from contact with bulk solvent.

3D domain-swapped human cystatin C with amyloidlike intermolecular beta-sheets.

Oligomerization of human cystatin C (HCC) leads to amyloid deposits in brain arteries, and this process is greatly accelerated with a naturally occurring L68Q variant. The crystal structures of N‐truncated and full‐length HCC (cubic form) showed dimer formation via three‐dimensional (3D) domain swapping, and this observation has led to the suggestion that an analogous domain‐swapping mechanism, but propagated in an open‐ended fashion, could be the basis of HCC fibril formation. Here we report that full‐length HCC, when crystallized in a new, tetragonal form, dimerizes by swapping the same secondary structure elements but with a very different overall structure generated by the flexibility of the hinge linking the moveable elements. The β‐strands of the β‐cores of the two folding units of the present dimer are roughly parallel, while they formed an angle of about 100° in the previous two structures. The dimers pack around a crystallographic dyad by extending their molecular β‐sheets in an intermolecular context. At the other edge of the molecular β‐sheet, side‐chain–side‐chain hydrogen bonds propagate the β‐structure in the same direction. In consequence, a supramolecular crystal structure is generated, with all the β‐strands of the domain‐swapped dimers being perpendicular to one crystallographic direction. This observation is relevant to amyloid aggregation of HCC, as X‐ray diffraction studies of amyloid fibrils show them to have ordered, repeating structure, consistent with the so‐called cross‐β structure, in which extended polypeptide chains are perpendicular to the fiber axis and form infinite β‐sheets that are parallel to this axis. Proteins 2005.

The Role of Cystatin C in Cerebral Amyloid Angiopathy and Stroke: Cell Biology and Animal Models

A variant of the cysteine protease inhibitor, cystatin c, forms amyloid deposited in the cerebral vasculature of patients with hereditary cerebral hemorrhage with amyloidosis, icelandic type (hchwa‐i), leading to cerebral hemorrhages early in life. however, cystatin c is also implicated in neuronal degenerative diseases in which it does not form the amyloid protein, such as alzheimer disease (ad). accumulating data suggest involvement of cystatin c in the pathogenic processes leading to amyloid deposition in cerebral vasculature and most significantly to cerebral hemorrhage in patients with cerebral amyloid angiopathy (caa). This review focuses on cell culture and animal models used to study the role of cystatin c in these processes.