A Dance with Protein Assemblies : Analysis, Structure Prediction, and Design

Protein assemblies are some of the most complex molecular machines in nature. They facilitate many cellular functions, from DNA replication to molecular motion, energy production, and even the production of other proteins. In a series of 3 papers, we analyzed the structure, developed structure prediction tools, and design tools, for different protein assemblies. Many of the studies were centered around viral protein capsids. Viral capsids are protein coats found inside viruses that contain and protect the viral genome. In one paper, we studied the interfaces of these capids and their energy landscapes. We found that they differ from regular homomers in terms of the amino acid composition and size, but not in the quality of interactions. This contradicts existing experimental and theoretical studies that suggest that the interactions are weak. We hypothesise that the occlusion by our models of electrostatic and entropic contributions might be at play. In another paper, we developed methods to predict large cubic symmetrical protein assemblies, such as viral capsids, from sequence. This method is based upon AlphaFold, a new AI tool that has revolutionized protein structure prediction. We found that we can predict up to 50% of the structures of these assemblies. The method can quickly elucidate the structure of many relevant proteins for humans, and for understanding structures relevant to disease, such as the structures of viral capsids. In the final paper, we developed tools to design capsid-like proteins called cages – structures that can be used for drug delivery and vaccine design. A fundamental problem in designing cage structures is achieving different architectures and low porosity, goals that are important for vaccine design and the delivery of small drug molecules. By explicitly modelling the shapes of the subunits in the cage and matching the shapes with proteins from structural databases, we find that we can create structures with many different sizes, shapes, and porosities - including low porosities. While waiting for experimental validation, the design strategy described in the paper must be extended, and more designs must be tested

Novel Chemokine-Based Immunotoxins for Potent and Selective Targeting of Cytomegalovirus Infected Cells

Immunotoxins as antiviral therapeutics are largely unexplored but have promising prospective due to their high selectivity potential and their unparalleled efficiency. One recent example targeted the virus-encoded G protein-coupled receptor US28 as a strategy for specific and efficient treatment of human cytomegalovirus (HCMV) infections. US28 is expressed on virus-infected cells and scavenge chemokines by rapid internalization. The chemokine-based fusion-toxin protein (FTP) consisted of a variant (F49A) of CX3CL1 specifically targeting US28 linked to the catalytic domain of Pseudomonas exotoxin A (PE). Here, we systematically seek to improve F49A-FTP by modifications in its three structural domains; we generated variants with (1) altered chemokine sequence (K14A, F49L, and F49E), (2) shortened and elongated linker region, and (3) modified toxin domain. Only F49L-FTP displayed higher selectivity in its binding to US28 versus CX3CR1, the endogenous receptor for CX3CL1, but this was not matched by a more selective killing of US28-expressing cells. A longer linker and different toxin variants decreased US28 affinity and selective killing. Thereby, F49A-FTP represents the best candidate for HCMV treatment. Many viruses encode internalizing receptors suggesting that not only HCMV but also, for instance, Epstein-Barr virus and Kaposi’s sarcoma-associated herpesvirus may be targeted by FTPs

Crystal Structure of the Bovine Mitochondrial Elongation Factor Tu·Ts Complex

The three-dimensional structure of the bovine mitochondrial elongation factor (EF)-Tu.Ts complex (EF-Tumt.Tsmt) has been determined to 2.2-A resolution using the multi-wavelength anomalous dispersion experimental method. This complex provides the first insight into the structure of EF-Tsmt. EF-Tsmt is similar to Escherichia coli and Thermus thermophilus EF-Ts in the amino-terminal domain. However, the structure of EF-Tsmt deviates considerably in the core domain with a five-stranded beta-sheet forming a portion of subdomain N of the core. In E. coli EF-Ts, this region is composed of a three-stranded sheet. The coiled-coil domain of the E. coli EF-Ts is largely eroded in EF-Tsmt, in which it consists of a large loop packed against subdomain C of the core. The conformation of bovine EF-Tumt in complex with EF-Tsmt is distinct from its conformation in the EF-Tumt.GDP complex. When domain III of bovine EF-Tumt.GDP is superimposed on domain III of EF-Tumt in the EF-Tumt.Tsmt complex, helix B from domain I is also almost superimposed. However, the rest of domain I is rotated relative to this helix toward domain II, which itself is rotated toward domain I relative to domain III. Extensive contacts are observed between the amino-terminal domain of EF-Tsmt and domain I of EF-Tumt. Furthermore, the conserved TDFV sequence of EF-Tsmt also contacts domain I with the side chain of Asp139 contacting helix B of EF-Tumt and inserting the side chain of Phe140 between helices B and C. The structure of the EF-Tumt.Tsmt complex provides new insights into the nucleotide exchange mechanism and provides a framework for explaining much of the mutational data obtained for this complex

miRNA profiling of circulating EpCAM(+) extracellular vesicles:promising biomarkers of colorectal cancer

Cancer cells secrete small membranous extracellular vesicles (EVs) into their microenvironment and circulation. These contain biomolecules, including proteins and microRNAs (miRNAs). Both circulating EVs and miRNAs have received much attention as biomarker candidates for non-invasive diagnostics. Here we describe a sensitive analytical method for isolation and subsequent miRNA profiling of epithelial-derived EVs from blood samples of patients with colorectal cancer (CRC). The epithelial-derived EVs were isolated by immunoaffinity-capture using the epithelial cell adhesion molecule (EpCAM) as marker. This approach mitigates some of the specificity issues observed in earlier studies of circulating miRNAs, in particular the negative influence of miRNAs released by erythrocytes, platelets and non-epithelial cells. By applying this method to 2 small-scale patient cohorts, we showed that blood plasma isolated from CRC patients prior to surgery contained elevated levels of 13 EpCAM+-EV miRNAs compared with healthy individuals. Upon surgical tumour removal, the plasma levels of 8 of these were reduced (miR-16-5p, miR-23a-3p, miR-23b-3p, miR-27a-3p, miR-27b-3p, miR-30b-5p, miR-30c-5p and miR-222-3p). These findings indicate that the miRNAs are of tumour origin and may have potential as non-invasive biomarkers for detection of CRC. This work describes a non-invasive blood-based method for sensitive detection of cancer with potential for clinical use in relation to diagnosis and screening. We used the method to study CRC; however, it is not restricted to this disease. It may in principle be used to study any cancer that release epithelial-derived EVs into circulation

Rationally designed chemokine-based toxin targeting the viral G protein-coupled receptor US28 potently inhibits cytomegalovirus infection in vivo

The use of receptor–ligand interactions to direct toxins to kill diseased cells selectively has shown considerable promise for treatment of a number of cancers and, more recently, autoimmune disease. Here we move the fusion toxin protein (FTP) technology beyond cancer/autoimmune therapeutics to target the human viral pathogen, human cytomegalovirus (HCMV), on the basis of its expression of the 7TM G protein-coupled chemokine receptor US28. The virus origin of US28 provides an exceptional chemokine-binding profile with high selectivity and improved binding for the CX3C chemokine, CX(3)CL1. Moreover, US28 is constitutively internalizing by nature, providing highly effective FTP delivery. We designed a synthetic CX(3)CL1 variant engineered to have ultra-high affinity for US28 and greater specificity for US28 than the natural sole receptor for CX(3)CL1, CX(3)CR1, and we fused the synthetic variant with the cytotoxic domain of Pseudomonas Exotoxin A. This novel strategy of a rationally designed FTP provided unparalleled anti-HCMV efficacy and potency in vitro and in vivo