Optimizing NMR fragment-based drug screening for membrane protein targets.

NMR spectroscopy has played a pivotal role in fragment-based drug discovery by coupling detection of weak ligand-target binding with structural mapping of the binding site. Fragment-based screening by NMR has been successfully applied to many soluble protein targets, but only to a limited number of membrane proteins, despite the fact that many drug targets are membrane proteins. This is partly because of difficulties preparing membrane proteins for NMR-especially human membrane proteins-and because of the inherent complexity associated with solution NMR spectroscopy on membrane protein samples, which require the inclusion of membrane-mimetic agents such as micelles, nanodiscs, or bicelles. Here, we developed a generalizable protocol for fragment-based screening of membrane proteins using NMR. We employed two human membrane protein targets, both in fully protonated detergent micelles: the single-pass C-terminal domain of the amyloid precursor protein, C99, and the tetraspan peripheral myelin protein 22 (PMP22). For both we determined the optimal NMR acquisition parameters, protein concentration, protein-to-micelle ratio, and upper limit to the concentration of D6-DMSO in screening samples. Furthermore, we conducted preliminary screens of a plate-format molecular fragment mixture library using our optimized conditions and were able to identify hit compounds that selectively bound to the respective target proteins. It is hoped that the approaches presented here will be useful in complementing existing methods for discovering lead compounds that target membrane proteins.

Chain-Selective Isotopic Labeling of the Heterodimeric Type III Secretion Chaperone, Scc4:Scc1, Reveals the Total Structural Rearrangement of the Chlamydia trachomatis Bi-Functional Protein, Scc4.

Scc4 is an unusual bi-functional protein from Chlamydia trachomatis (CT) that functions as a type III secretion system (T3SS) chaperone and an RNA polymerase (RNAP)-binding protein. Both functions require interactions with protein partners during specific stages of the CT developmental cycle. As a T3SS chaperone, Scc4 binds Scc1 during the late stage of development to form a heterodimer complex, which chaperones the essential virulence effector, CopN. During the early-middle stage of development, Scc4 regulates T3SS gene expression by binding the σ66-containing RNAP holoenzyme. In order to study the structure and association mechanism of the Scc4:Scc1 T3SS chaperone complex using nuclear magnetic resonance (NMR) spectroscopy, we developed an approach to selectively label each chain of the Scc4:Scc1 complex with the 15N-isotope. The approach allowed one protein to be visible in the NMR spectrum at a time, which greatly reduced resonance overlap and permitted comparison of the backbone structures of free and bound Scc4. 1H,15N-heteronuclear single quantum coherence spectra of the 15N-Scc4:Scc1 and Scc4:15N-Scc1 complexes showed a total structural rearrangement of Scc4 upon binding Scc1 and a dynamic region isolated to Scc1, respectively. Development of the chain-selective labeling approach revealed that the association of Scc4 and Scc1 requires partial denaturation of Scc1 to form the high affinity complex, while low affinity interactions occurred between the isolated proteins under non-denaturing conditions. These results provide new models for Scc4's functional switching mechanism and Scc4:Scc1 association in CT.

Backbone and sidechain resonance assignments and secondary structure of Scc4 from Chlamydia trachomatis.

Chlamydia trachomatis is an obligate intracellular bacterium that causes the most common sexually transmitted bacterial diseases in the world. With a biphasic developmental cycle, the bacteria utilize a type III secretion system (T3SS) to invade host cells as infectious elemental bodies, which then differentiate into actively dividing reticulate bodies. The regulation of the developmental cycle and the T3SS are linked by the bi-functional protein, specific Chlamydia chaperone 4 (Scc4). Scc4 is a class I T3SS chaperone forming a heterodimer with specific Chlamydia chaperone 1 (Scc1) to chaperone the essential virulence effector, Chlamydia outer membrane protein N. Scc4 also functions as a transcription factor by binding to the RNA polymerase holoenzyme between the flap region of the ß subunit and region 4 of σ66. In order to investigate the mechanism behind Scc4's dual functions and target its protein-protein interactions as a route for drug development, the structure and dynamics of Scc4 are being pursued. In the course of this effort, we assigned 89.2% of the backbone and sidechain 1H, 15N, and 13C resonances of full-length Scc4. The assigned chemical shifts were used to predict the secondary structure and dynamic properties. The type and order of Scc4's determined secondary structure are consistent with the X-ray crystal structures of other bacterial T3SS chaperones.

Purification of Tag-Free Chlamydia trachomatis Scc4 for Structural Studies Using Sarkosyl-Assisted on-Column Complex Dissociation.

Chlamydia trachomatis is an obligate intracellular bacterial pathogen that causes the most common sexually transmitted bacterial disease in the world. The bacterium has a unique biphasic developmental cycle with a type III secretion system (T3SS) to invade host cells. Scc4 is a class I T3SS chaperone forming a heterodimer complex with Scc1 to chaperone the essential virulence effector, CopN. Scc4 also functions as an RNA polymerase binding protein to regulate σ66-dependent transcription. Aggregation and low solubility of 6X-histidine-tagged Scc4 and the insolubility of 6X-histidine and FLAG-tagged Scc1 expressed in Escherichia coli have hindered the high-resolution nuclear magnetic resonance (NMR) structure determination of these proteins and motivated the development of an on-column complex dissociation method to produce tag-free Scc4 and soluble FLAG-tagged Scc1. By utilizing a 6X-histidine-tag on one protein, the coexpressed Scc4-Scc1 complex was captured on nickel-charged immobilized metal affinity chromatography resin, and the nondenaturing detergent, sodium N-lauroylsarcosine (sarkosyl), was used to dissociate and elute the non-6X-histidine-tagged protein. Tag-free Scc4 was produced in a higher yield and had better NMR spectral characteristics compared to 6X-histidine-tagged Scc4, and soluble FLAG-tagged Scc1 was purified for the first time in a high yield. The backbone structure of Scc4 after exposure to sarkosyl was validated using NMR spectroscopy, demonstrating the usefulness of the method to produce proteins for structural and functional studies. The sarkosyl-assisted on-column complex dissociation method is generally applicable to protein complexes with high affinity and is particularly useful when affinity tags alter the protein's biophysical properties or when coexpression is necessary for solubility.