Structure-based discovery of fiber-binding compounds that reduce the cytotoxicity of amyloid beta.

Amyloid protein aggregates are associated with dozens of devastating diseases including Alzheimer's, Parkinson's, ALS, and diabetes type 2. While structure-based discovery of compounds has been effective in combating numerous infectious and metabolic diseases, ignorance of amyloid structure has hindered similar approaches to amyloid disease. Here we show that knowledge of the atomic structure of one of the adhesive, steric-zipper segments of the amyloid-beta (Aβ) protein of Alzheimer's disease, when coupled with computational methods, identifies eight diverse but mainly flat compounds and three compound derivatives that reduce Aβ cytotoxicity against mammalian cells by up to 90%. Although these compounds bind to Aβ fibers, they do not reduce fiber formation of Aβ. Structure-activity relationship studies of the fiber-binding compounds and their derivatives suggest that compound binding increases fiber stability and decreases fiber toxicity, perhaps by shifting the equilibrium of Aβ from oligomers to fibers. DOI:http://dx.doi.org/10.7554/eLife.00857.001

Structure of thymidylate kinase from Ehrlichia chaffeensis

A 2.15 Å resolution apo structure of thymidylate kinase from E. chaffeensis is reported

Immobilized metal-affinity chromatography protein-recovery screening is predictive of crystallographic structure success

An overview of the methods used for high-throughput cloning and protein-expression screening of SSGCID hexahistidine recombinant proteins is provided. It is demonstrated that screening for recombinant proteins that are highly recoverable from immobilized metal-affinity chromatography improves the likelihood that a protein will produce a structure

High-throughput protein production and purification at the Seattle Structural Genomics Center for Infectious Disease

An overview of the standard SSGCID protein-purification protocol is given and success rates and cleavage alternatives are discussed

Structure of the cystathionine γ-synthase MetB from Mycobacterium ulcerans

Cystathionine γ-synthase (CGS) is a transferase that catalyzes the reaction between O 4-succinyl-l-homoserine and l-cysteine to produce l-­cystathionine and succinate. The crystal structure of CGS from M. ulcerans is presented covalently linked to the cofactor pyridoxal phosphate (PLP). A second structure contains PLP as well as a highly ordered HEPES molecule in the active site acting as a pseudo-ligand. This is the first structure ever reported from the pathogen M. ulcerans

Structure of a Burkholderia pseudomallei Trimeric Autotransporter Adhesin Head

Pathogenic bacteria adhere to the host cell surface using a family of outer membrane proteins called Trimeric Autotransporter Adhesins (TAAs). Although TAAs are highly divergent in sequence and domain structure, they are all conceptually comprised of a C-terminal membrane anchoring domain and an N-terminal passenger domain. Passenger domains consist of a secretion sequence, a head region that facilitates binding to the host cell surface, and a stalk region.Pathogenic species of Burkholderia contain an overabundance of TAAs, some of which have been shown to elicit an immune response in the host. To understand the structural basis for host cell adhesion, we solved a 1.35 A resolution crystal structure of a BpaA TAA head domain from Burkholderia pseudomallei, the pathogen that causes melioidosis. The structure reveals a novel fold of an intricately intertwined trimer. The BpaA head is composed of structural elements that have been observed in other TAA head structures as well as several elements of previously unknown structure predicted from low sequence homology between TAAs. These elements are typically up to 40 amino acids long and are not domains, but rather modular structural elements that may be duplicated or omitted through evolution, creating molecular diversity among TAAs.The modular nature of BpaA, as demonstrated by its head domain crystal structure, and of TAAs in general provides insights into evolution of pathogen-host adhesion and may provide an avenue for diagnostics

Design, Characterization and Applications of Symmetric Protein Scaffolds A

Proteins are essential macromolecules for all living organisms. They provide cellularstructure and perform most of the metabolic functions essential for all life. The importance ofproteins makes them the most studied and exploited macromolecules. We can exploit thestructure of a protein to design a specific therapeutic to treat a disease or we can use proteins as biocatalysts for the efficient creation of molecules. In many instances, these applications of proteins are difficult to achieve. The work in this dissertation focuses on the development and evaluations of novel techniques to aid in the study and use of proteins.The first part of this dissertation focus on the creation of a series of symmetric oligomersto be used as crystallization scaffolds. Such scaffolds are intended to induce their symmetry onto asymmetric protein crystallization target proteins. The ability to determine the crystal structure can be essential for the creation of new targeted drugs or the better understanding of a biological process. Unfortunately many proteins fail to crystallize for reasons that are not well understood. It is thought that such induction of symmetry and variety of geometrically distinct scaffolds will aid in the crystallization of difficult-to-crystallize proteins. Preliminary results of these novel scaffolds and existing scaffolds are described.In the second part, applications of symmetric scaffolds for the creation of enzymatic materials are presented. These purely proteinaceous assemblies are designed to replicate previousdescribed enzyme encapsulating materials. These materials typically improve enzyme reaction rates and product extraction. The final part of the dissertation focuses on the shell protein PduA from the 1,2-propanediol-utilization bacterial microcompartment (MCPs). These MCPs encapsulate metabolic pathways and contain volatile or toxic pathway intermediates. Research into turning these MCPs into bioreactors containing non-native enzymes is ongoing in many labs. Full realization of this technology relies on the encapsulation of new metabolic enzymes and transport of novel substrate and products through the shells. These processes are poorly understood, here structural studies of shell protein permutations. These permutations alter the topology of the shell protein allowing the scaffolding of proteins to the exterior surface of the MCPs. Finally, the efforts to elicited the interaction of specific targeting sequences to shell protein by x-ray crystallography are discussed

Structure-based discovery of fiber-binding compounds that reduce the cytotoxicity of amyloid beta.

Amyloid protein aggregates are associated with dozens of devastating diseases including Alzheimer's, Parkinson's, ALS, and diabetes type 2. While structure-based discovery of compounds has been effective in combating numerous infectious and metabolic diseases, ignorance of amyloid structure has hindered similar approaches to amyloid disease. Here we show that knowledge of the atomic structure of one of the adhesive, steric-zipper segments of the amyloid-beta (Aβ) protein of Alzheimer's disease, when coupled with computational methods, identifies eight diverse but mainly flat compounds and three compound derivatives that reduce Aβ cytotoxicity against mammalian cells by up to 90%. Although these compounds bind to Aβ fibers, they do not reduce fiber formation of Aβ. Structure-activity relationship studies of the fiber-binding compounds and their derivatives suggest that compound binding increases fiber stability and decreases fiber toxicity, perhaps by shifting the equilibrium of Aβ from oligomers to fibers. DOI:http://dx.doi.org/10.7554/eLife.00857.001