Chlamydia protein Pgp3 studied at high resolution in a new crystal form

The protein Pgp3 is implicated in the sexually transmitted disease chlamydia and comprises an extended complex arrangement of a C terminal domain (CTD) and an N terminal domain (NTD), each linked by a triple helix coiled coil (THCC). We report the X-ray crystal structure of Pgp3 from a LGV1 strain at the highest X-ray diffraction resolution obtained to date for the full protein. The protein was crystallised using a high KBr salt concentration, which resulted in a new crystal form with relatively low solvent content diffracting to a resolution of 1.98 Å. We describe the 3D structure of this new crystal form, compare it with other crystal forms, describe the KBr salt binding sites and the relevance to chlamydia isolates from around the globe. The crystal packing is apparently driven by the CTDs. Since the three fold axes of the THCC and NTD are not collinear with a CTD’s three fold axis this naturally leads to a disorder in the THCC and the portion of the NTD not directly interacting with the CTD via crystal packing. The key avenue to resolve these oddities of the crystal structure analysis was a complete new analysis in space group P1 and determining the space group as P212121. This space group assignment was the one originally determined from the diffraction pattern but perhaps complicated by a translational non crystallographic symmetry. We found this crystal structure of a three domain multi macromolecular complex, with two misaligned three fold axes, a unique challenge, something not encountered before. A specific intermolecular interaction, possibly of functional significance in receptor binding in chlamydia, we suggest might allow design of a new chemotherapeutic agent against chlamydia

A Linear Epitope in the N-Terminal Domain of CCR5 and Its Interaction with Antibody.

The CCR5 receptor plays a role in several key physiological and pathological processes and is an important therapeutic target. Inhibition of the CCR5 axis by passive or active immunisation offers one very selective strategy for intervention. In this study we define a new linear epitope within the extracellular domain of CCR5 recognised by two independently produced monoclonal antibodies. A short peptide encoding the linear epitope can induce antibodies which recognise the intact receptor when administered colinear with a tetanus toxoid helper T cell epitope. The monoclonal antibody RoAb 13 is shown to bind to both cells and peptide with moderate to high affinity (6x10^8 and 1.2x107 M-1 respectively), and binding to the peptide is enhanced by sulfation of tyrosines at positions 10 and 14. RoAb13, which has previously been shown to block HIV infection, also blocks migration of monocytes in response to CCR5 binding chemokines and to inflammatory macrophage conditioned medium. A Fab fragment of RoAb13 has been crystallised and a structure of the antibody is reported to 2.1 angstrom resolution

Improved Success of Sparse Matrix Protein Crystallization Screening with Heterogeneous Nucleating Agents

Crystallization is a major bottleneck in the process of macromolecular structure determination by X-ray crystallography. Successful crystallization requires the formation of nuclei and their subsequent growth to crystals of suitable size. Crystal growth generally occurs spontaneously in a supersaturated solution as a result of homogenous nucleation. However, in a typical sparse matrix screening experiment, precipitant and protein concentration are not sampled extensively, and supersaturation conditions suitable for nucleation are often missed

Attenuated total reflection-FT-IR spectroscopic imaging of protein crystallization

Protein crystallization is of strategic and commercial relevance in the post-genomic era because of its pivotal role in structural proteomics projects. Although protein structures are crucial for understanding the function of proteins and to the success of rational drug design and other biotechnology applications, obtaining high quality crystals is a major bottleneck to progress. The major means of obtaining crystals is by massive-scale screening of a target protein solution with numerous crystallizing agents. However, when crystals appear in these screens, one cannot easily know if they are crystals of protein, salt, or any other molecule that happens to be present in the trials. We present here a method based on Attenuated Total Reflection (ATR)-FT-IR imaging that reliably identifies protein crystals through a combination of chemical specificity and the visualizing capability of this approach, thus solving a major hurdle in protein crystallization. ATR-FT-IR imaging was successfully applied to study the crystallization of thaumatin and lysozyme in a high-throughput manner, simultaneously from six different solutions. This approach is fast as it studies protein crystallization in situ and provides an opportunity to examine many different samples under a range of conditions

Trends and Challenges in Experimental Macromolecular Crystallography

Macromolecular X-ray crystallography underpins the vigorous field of structural molecular biology having yielded many protein, nucleic acid and virus structures in fine detail. The understanding of the recognition by these macromolecules, as receptors, of their cognate ligands involves the detailed study of the structural chemistry of their molecular interactions. Also these structural details underpin the rational design of novel inhibitors in modern drug discovery in the pharmaceutical industry. Moreover, from such structures the functional details can be inferred, such as the biological chemistry of enzyme reactivity. There is then a vast number and range of types of biological macromolecules that potentially could be studied. The completion of the protein primary sequencing of the yeast genome, and the human genome sequencing project comprising some 105 proteins that is underway, raises expectations for equivalent three dimensional structural database