Structural and biophysical properties of FopA, a major outer membrane protein of Francisella tularensis.

Francisella tularensis is an extremely infectious pathogen and a category A bioterrorism agent. It causes the highly contagious zoonosis, Tularemia. Currently, FDA approved vaccines against tularemia are unavailable. F. tularensis outer membrane protein A (FopA) is a well-studied virulence determinant and protective antigen against tularemia. It is a major outer membrane protein (Omp) of F. tularensis. However, FopA-based therapeutic intervention is hindered due to lack of complete structural information for membrane localized mature FopA. In our study, we established recombinant expression, monodisperse purification, crystallization and X-ray diffraction (~6.5 Å) of membrane localized mature FopA. Further, we performed bioinformatics and biophysical experiments to unveil its structural organization in the outer membrane. FopA consists of 393 amino acids and has less than 40% sequence identity to known bacterial Omps. Using comprehensive sequence alignments and structure predictions together with existing partial structural information, we propose a two-domain organization for FopA. Circular dichroism spectroscopy and heat modifiability assay confirmed FopA has a ß-barrel domain consistent with alphafold2's prediction of an eight stranded ß-barrel at the N-terminus. Small angle X-ray scattering (SAXS) and native-polyacrylamide gel electrophoresis revealed FopA purified in detergent micelles is predominantly dimeric. Molecular density derived from SAXS at 31 Å shows putative dimeric N-terminal ß-barrels surrounded by detergent corona and connected to C-terminal domains via flexible linker. Disorder analysis predicts N- and C-terminal domains are interspersed by a long intrinsically disordered region and alphafold2 predicts this region to be largely unstructured. Taken together, we propose a dimeric, two-domain organization of FopA in the outer membrane: the N-terminal ß-barrel is membrane embedded, provides dimerization interface and tethers to membrane extrinsic C-terminal domain via long flexible linker. Structure determination of membrane localized mature FopA is essential to understand its role in pathogenesis and develop anti-tularemia therapeutics. Our results pave the way towards it.

Purification, characterization, and preliminary serial crystallography diffraction advances structure determination of full-length human particulate guanylyl cyclase A receptor.

Particulate Guanylyl Cyclase Receptor A (pGC-A) is a natriuretic peptide membrane receptor, playing a vital role in controlling cardiovascular, renal, and endocrine functions. The extracellular domain interacts with natriuretic peptides and triggers the intracellular guanylyl cyclase domain to convert GTP to cGMP. To effectively develop methods to regulate pGC-A, structural information on the full-length form is needed. However, structural data on the transmembrane and intracellular domains are lacking. This work presents expression and optimization using baculovirus, along with the first purification of functional full-length human pGC-A. In vitro assays revealed the pGC-A tetramer was functional in detergent micelle solution. Based on our purification results and previous findings that dimer formation is required for functionality, we propose a tetramer complex model with two functional subunits. Previous research suggested pGC-A signal transduction is an ATP-dependent, two-step mechanism. Our results show the binding ligand also moderately activates pGC-A, and ATP is not crucial for activation of guanylyl cyclase. Furthermore, crystallization of full-length pGC-A was achieved, toward determination of its structure. Needle-shaped crystals with 3 Å diffraction were observed by serial crystallography. This work paves the road for determination of the full-length pGC-A structure and provides new information on the signal transduction mechanism.

Characterization and computational simulation of human Syx, a RhoGEF implicated in glioblastoma.

Structural discovery of guanine nucleotide exchange factor (GEF) protein complexes is likely to become increasingly relevant with the development of new therapeutics targeting small GTPases and development of new classes of small molecules that inhibit protein-protein interactions. Syx (also known as PLEKHG5 in humans) is a RhoA GEF implicated in the pathology of glioblastoma (GBM). Here we investigated protein expression and purification of ten different human Syx constructs and performed biophysical characterizations and computational studies that provide insights into why expression of this protein was previously intractable. We show that human Syx can be expressed and isolated and Syx is folded as observed by circular dichroism (CD) spectroscopy and actively binds to RhoA as determined by co-elution during size exclusion chromatography (SEC). This characterization may provide critical insights into the expression and purification of other recalcitrant members of the large class of oncogenic-Diffuse B-cell lymphoma (Dbl) homology GEF proteins. In addition, we performed detailed homology modeling and molecular dynamics simulations on the surface of a physiologically realistic membrane. These simulations reveal novel insights into GEF activity and allosteric modulation by the plekstrin homology (PH) domain. These newly revealed interactions between the GEF PH domain and the membrane embedded region of RhoA support previously unexplained experimental findings regarding the allosteric effects of the PH domain from numerous activity studies of Dbl homology GEF proteins. This work establishes new hypotheses for structural interactivity and allosteric signal modulation in Dbl homology RhoGEFs.

Co-flow injection for serial crystallography at X-ray free-electron lasers.

Serial femtosecond crystallography (SFX) is a powerful technique that exploits X-ray free-electron lasers to determine the structure of macro-molecules at room temperature. Despite the impressive exposition of structural details with this novel crystallographic approach, the methods currently available to introduce crystals into the path of the X-ray beam sometimes exhibit serious drawbacks. Samples requiring liquid injection of crystal slurries consume large quantities of crystals (at times up to a gram of protein per data set), may not be compatible with vacuum configurations on beamlines or provide a high background due to additional sheathing liquids present during the injection. Proposed and characterized here is the use of an immiscible inert oil phase to supplement the flow of sample in a hybrid microfluidic 3D-printed co-flow device. Co-flow generation is reported with sample and oil phases flowing in parallel, resulting in stable injection conditions for two different resin materials experimentally. A numerical model is presented that adequately predicts these flow-rate conditions. The co-flow generating devices reduce crystal clogging effects, have the potential to conserve protein crystal samples up to 95% and will allow degradation-free light-induced time-resolved SFX.

Biophysical characterization and a roadmap towards the NMR solution structure of G0S2, a key enzyme in non-alcoholic fatty liver disease.

In the United States non-alcoholic fatty liver disease (NAFLD) is the most common form of chronic liver disease, affecting an estimated 80 to 100 million people. It occurs in every age group, but predominantly in people with risk factors such as obesity and type 2 diabetes. NAFLD is marked by fat accumulation in the liver leading to liver inflammation, which may lead to scarring and irreversible damage progressing to cirrhosis and liver failure. In animal models, genetic ablation of the protein G0S2 leads to alleviation of liver damage and insulin resistance in high fat diets. The research presented in this paper aims to aid in rational based drug design for the treatment of NAFLD by providing a pathway for a solution state NMR structure of G0S2. Here we describe the expression of G0S2 in an E. coli system from two different constructs, both of which are confirmed to be functionally active based on the ability to inhibit the activity of Adipose Triglyceride Lipase. In one of the constructs, preliminary NMR spectroscopy measurements show dominant alpha-helical characteristics as well as resonance assignments on the N-terminus of G0S2, allowing for further NMR work with this protein. Additionally, the characterization of G0S2 oligomers are outlined for both constructs, suggesting that G0S2 may defensively exist in a multimeric state to protect and potentially stabilize the small 104 amino acid protein within the cell. This information presented on the structure of G0S2 will further guide future development in the therapy for NAFLD.

Protein expression and purification of G-protein coupled receptor kinase 6 (GRK6), toward structure-based drug design and discovery for multiple myeloma.

Human G-protein coupled receptor kinase 6 (GRK6) belongs to the GRK4 kinase subfamily of the G protein-coupled receptor kinase family which comprises of GRK1, GRK2, and GRK4. These kinases phosphorylate ligand-activated G-protein coupled receptors (GPCRs), driving heterotrimeric G protein coupling, desensitization of GPCR, and ß-arrestin recruitment. This reaction series mediates cellular signal pathways for cell survival, proliferation, migration and chemotaxis. GRK6 is a kinase target in multiple myeloma since it is highly expressed in myeloma cells compared to epithelial cells and has a significant role in mediating the chemotactic responses of T and B-lymphocytes. To support structure-based drug design, we describe three human GRK6 constructs, GRK6, GRK6His/EK, and GRK6His/TEV, designed for protein expression in Spodoptera frugiperda Sf9 insect cells. The first construct did not contain any purification tag whereas the other two constructs contained the His10 affinity tag, which increased purification yields. We report here that all three constructs of GRK6 were overexpressed in Sf9 insect cells and purified to homogeneity at levels that were suitable for co-crystallization of GRK6 with potential inhibitors. The yields of purified GRK6, GRK6His/EK, and GRK6His/TEV were 0.3 mg, 0.8 mg and 0.7 mg per liter of cell culture, respectively. In addition, we have shown that GRK6His/TEV with the His10 tag removed was highly homogeneous and monodisperse as observed by dynamic light scattering measurement and actively folded as exhibited by circular dichroism spectroscopy. The described methods will support the structure-based development of additional therapeutics against multiple myeloma.

Structural enzymology using X-ray free electron lasers.

Mix-and-inject serial crystallography (MISC) is a technique designed to image enzyme catalyzed reactions in which small protein crystals are mixed with a substrate just prior to being probed by an X-ray pulse. This approach offers several advantages over flow cell studies. It provides (i) room temperature structures at near atomic resolution, (ii) time resolution ranging from microseconds to seconds, and (iii) convenient reaction initiation. It outruns radiation damage by using femtosecond X-ray pulses allowing damage and chemistry to be separated. Here, we demonstrate that MISC is feasible at an X-ray free electron laser by studying the reaction of M. tuberculosis ß-lactamase microcrystals with ceftriaxone antibiotic solution. Electron density maps of the apo-ß-lactamase and of the ceftriaxone bound form were obtained at 2.8 Å and 2.4 Å resolution, respectively. These results pave the way to study cyclic and non-cyclic reactions and represent a new field of time-resolved structural dynamics for numerous substrate-triggered biological reactions.

Full inactivation of alphaviruses in single particle and crystallized forms.

Inherent in the study of viruses is the risk of pathogenic exposure, which necessitates appropriate levels of biosafety containment. Unfortunately, this also limits the availability of useful research instruments that are located at facilities not equipped to handle infectious pathogens. Abrogation of viral infectivity can be accomplished without severely disrupting the physical structure of the virus particle. Virus samples that are verifiably intact but not infectious may be enabled for study at research facilities where they would otherwise not be allowed. Inactivated viruses are also used in the development of vaccines, where immunogenicity is sought in the absence of active infection. We demonstrate the inactivation of Sindbis alphavirus particles in solution, as well as in crystallized form. Inactivation was accomplished by two different approaches: crosslinking of proteins by glutaraldehyde treatment, and crosslinking of nucleic acids by UV irradiation. Biophysical characterization methods, including dynamic light scattering and transmission electron microscopy, were used to demonstrate that the glutaraldehyde and UV inactivated Sindbis virus particles remain intact structurally. SDS-PAGE was also used to show evidence of the protein crosslinking that was expected with glutaraldehyde treatment, but also observed with UV irradiation.

High-resolution NMR reveals secondary structure and folding of amino acid transporter from outer chloroplast membrane.

Solving high-resolution structures for membrane proteins continues to be a daunting challenge in the structural biology community. In this study we report our high-resolution NMR results for a transmembrane protein, outer envelope protein of molar mass 16 kDa (OEP16), an amino acid transporter from the outer membrane of chloroplasts. Three-dimensional, high-resolution NMR experiments on the (13)C, (15)N, (2)H-triply-labeled protein were used to assign protein backbone resonances and to obtain secondary structure information. The results yield over 95% assignment of N, HN, CO, Cα, and Cß chemical shifts, which is essential for obtaining a high resolution structure from NMR data. Chemical shift analysis from the assignment data reveals experimental evidence for the first time on the location of the secondary structure elements on a per residue basis. In addition T 1Z and T2 relaxation experiments were performed in order to better understand the protein dynamics. Arginine titration experiments yield an insight into the amino acid residues responsible for protein transporter function. The results provide the necessary basis for high-resolution structural determination of this important plant membrane protein.