Crystallographic evidence for oxidative Met→Asp conversion in human hemoglobin

The mutants HbA Bristol-Alesha (βV(E11)67M) and HbF Toms River (γV(E11)67M) [1,2] are examples of a `silent' posttranslational modification in which the side chain of the substituted amino acid is chemically modified (Met→Asp) resulting in a disparity between the DNA and protein sequences. In both cases the patients' hemolysate contained both V67M and V67D isoforms. But in the analogous α subunit mutant, Hb Evans αV(E11)62M, the conversion to Asp was not identified and DNA sequencing confirmed the Met replacement [3]. Our crystal structures of the three (ferrous) CO-bound recombinant V(E11)M mutants show the MetE11 side chain in similar conformations. But the air-oxidized β mutant crystals clearly showed a `bifurcated' and smaller electron density pattern for the E11 side chain, indicating the appearance of Asp. Also, the ligand electron-density at the iron atom in the oxidized β subunit appears to be an oxoferryl Fe4+=O rather than a Fe3+OH2 ferric complex. In contrast, there was little change in the electron density for αMetE11 in oxidized αV62M crystals. The ligand in the ferric α subunit is clearly a coordinated water molecule. But again, a ferryl Fe4+=O complex appears to occur in the wild-type β subunit. This strongly suggest that β subunits have a greater propensity to form highly reactive ferryl species, and that the ferryl species play a role in the Met→Asp conversion. Our autoxidation and proteomics studies showed that although all three recombinant VE11M mutants had similar, high rates of autooxidation and a strong H2O2 dose dependence on sulfoxide and sulfone formation, no Asp formation was detected in α subunits whereas MetE11 is converted to Asp to levels as high as 15% in vitro in β and γ subunits. We propose that the Met→Asp conversion specifically involves H2O2 mediated oxidation of the ferrous heme to an oxoferryl state, and because the transient ferryl intermediates are much less stable in the α subunits, there is no oxidative conversion.

Sulfur-SAD phasing and UV-RIP analysis on a single Cysteine bridge protein

The advantage of using the anomalous signal of sulfur for phase determination is that only a single, well-diffracting crystal is needed and that a native structure will be obtained. Using long-wavelength S-SAD to a resolution of 1.9 Å we have determined the novel structure of an 89 residue protein with only 2 Cysteines fixed in a disulfide bridge. To the best of our knowledge, the Bijvoet ratio in our example is one of the smallest for which a successful structure solution by S-SAD has been reported. Data were collected on 3 different volumes of a single crystal at beamline 14.1. at BESSY II, Berlin [1], at a wavelength of 1.8 Å. At this wavelength the maximum resolution obtainable was 1.9 Å. The data were processed in space group I222 with a low resolution R-factor of 3.2% and a multiplicity of 17. Based on an anomalous correlation coefficient cut off at 30% the signal extends to 2.6 Å. The sulfur substructure was determined using AutoSol/HYSS [2] showing a total of four clear sulfur positions in the asymmetric unit with a resulting FOM of 0.27 and a BAYES Coefficient of 0.36. The crystal has a solvent content of 62% and the structure reveals a dimer and large solvent channels. Density modification lead to well-defined electron density maps for the protein and associated solvent molecules. This example demonstrates that S-SAD phase determination can work with as little as one S-atom per 45 amino acid residues. Additionally, we performed a UV-RIP (ultraviolet radiation damage-induced phasing) experiment in which a dataset was collected before and after irradiating the crystal with a hard UV laser. An isomorphous difference map shows the clear disruption of both disulfide bridges and we are currently working on combined phasing using both anomalous and isomorphous differences based on the S-SAD and UV-RIP data.

Crystallographic fragment screening: challenges, opportunities, lessons learned

Fragment-based approaches are now routinely applied for lead development in pharmaceutical drug research. Usually, a small but well selected library of low molecular weight compounds is pre-screened by biochemical or biophysical methods such as surface plasmon resonance (SPR), nuclear magnetic resonance (NMR) or thermal shift assay; often followed for promising hit candidates by X-ray crystallography. We designed a small fragment library consisting of 364 compounds that is not strictly compliant to the otherwise often followed Astex rule of three for fragment library composition.[1] Thereafter, our library was validated on the pepsin-like aspartyl protease endothiapepsin, which serves as a model system for proteins that are involved in serious diseases such as malaria (plasmepsins), hypertension (renin) and Alzheimer's disease (ß-secretase) and therefore, is a valid target for further drug development. Due to the small size of fragments, they frequently exhibit only low affinity to the applied target protein and thus are often hard to detect in any screening approach, reflected in little overlap between different screening methods. After initial screening, we decided to validate the entire library by X-ray crystallography, which requires a steady supply of crystals, reproducible soaking conditions and a reliable setup at a synchrotron source, such as HZB BESSY II BL14.1 [2], preferably with some automation in initial data processing and refinement. A total hit rate greater than 10% was obtained, which will be compared to results from other screening methods. The resulting crystal structures will be discussed and provide an ideal basis for further lead development.

Fragment-screening at the BESSY II MX-beamlines of the Helmholtz-Zentrum Berlin

"Within the last decade, the fragment-based screening approach has been matured to a reliable and powerful instrument of pharmaceutical drug discovery. The success of fragment screening strongly depends on the quality of the chosen fragment library (100-200 Da), the quality of the target protein diffraction as well as the possibility to use high throughput methods for the screen application. A thorough crystallographic analysis of many protein-fragment complex structures and their binding modes has the perspective to result in the development of new potential lead structures and to map the interaction landscape of protein surfaces. Recently we started the development of a dedicated experimental facility for high throughput fragment screening at the BESSY II storage ring. The in house data processing pipeline ""XDSAPP"" [1] has been developed to speed up the data evaluation of large amounts of diffraction data. We have assembled a fragment library of 96 compounds and have validated this library against two target proteins. These first results suggest that our library is capable of identifying binding partners at a hit rate of close to 10%. This library together with a fully automated beam line [2] will be made accessible to users, thus enabling fragment-screening experiments on a much broader basis. "

Facilities for Macromolecular Crystallography at the HZB

The Macromolecular Crystallography (MX) group at the Helmholtz-Zentrum Berlin (HZB) has been in operation since 2003. Since then, three state-of-the-art synchrotron beam lines (BL14.1-3) for MX have been built up on a 7T-wavelength shifter source [1,2]. Currently, the three beam lines represent the most productive MX-stations in Germany, with more than 1100 PDB depositions (Status 02/2014). BLs14.1 and 14.2 are energy tuneable in the range 5.5-15.5 keV, while beam line 14.3 is a fixed-energy side station operated at 13.8 keV. All three beam lines are equipped with state-of-the-art detectors: BL14.1 with a PILATUS 6M detector and BLs14.2 and 14.3 with large CCD-detectors. BL14.1 and BL14.2 are in regular user operation providing about 200 beam days per year and about 600 user shifts to approximately 70 research groups across Europe. BL14.3 has been equipped with a HC1 crystal dehydration device in 2011. In addition to serving the user community mainly as a screening and test beam line, it is currently the only MX beamline in Europe with a HC1 device permanently installed. Additional user facilities include office space adjacent to the beam lines, a sample preparation laboratory, a biology laboratory (safety level 1) and high-end computing resources. On the poster, a summary on the experimental possibilities of the beam lines and the ancillary equipment provided to the user community will be given.