Supraspinatus-to-Glenoid Contact Occurs During Standardized Overhead Reaching Motion

Background: Rotator cuff tears may result from repeated mechanical deformation of the cuff tendons, and internal impingement of the supraspinatus tendon against the glenoid is one such proposed mechanism of deformation. Purpose: To (1) describe the changing proximity of the supraspinatus tendon to the glenoid during a simulated overhead reaching task and (2) determine the relationship between scapular morphology and this proximity. Additionally, the patterns of supraspinatus-to-glenoid proximity were compared with previously described patterns of supraspinatus-to-coracoacromial arch proximity. Study Design: Descriptive laboratory study. Methods: Shoulder models were created from magnetic resonance images of 20 participants. Standardized kinematics were imposed on the models to simulate functional reaching, and the minimum distances between the supraspinatus tendon and the glenoid and the supraspinatus footprint and the glenoid were calculated every 5° between 0° and 150° of humerothoracic elevation. The angle at which contact between the supraspinatus and the glenoid occurred was documented. Additionally, the relationship between glenoid morphology (version and inclination) and the contact angle was evaluated. Descriptive statistics were calculated for the minimum distances, and glenoid morphology was assessed using Pearson correlation coefficients and simple linear regressions. Results: The minimum distances between the tendon and the glenoid and between the footprint and the glenoid decreased as elevation increased. Contact between the tendon and the glenoid occurred in all participant models at a mean elevation of 123° ± 10°. Contact between the footprint and the glenoid occurred in 13 of 20 models at a mean of 139° ± 10°. Less glenoid retroversion was associated with lower tendon-to-glenoid contact angles (r = -0.76; R (2) = 0.58; P \u3c .01). Conclusion: This study found that the supraspinatus tendon progressively approximated the glenoid during simulated overhead reaching. Additionally, all participant models eventually made contact with the glenoid by 150° of humerothoracic elevation, although anatomic factors influenced the precise angle at which contact occurred. Clinical Relevance: Contact between the supraspinatus and the glenoid may occur frequently within the range of elevation required for overhead activities. Therefore, internal impingement may be a prevalent mechanism for rotator cuff deformation that could contribute to cuff pathology

Structural Insight into How Bacteria Prevent Interference between Multiple Divergent Type IV Secretion Systems

Prokaryotes use type IV secretion systems (T4SSs) to translocate substrates (e.g., nucleoprotein, DNA, and protein) and/or elaborate surface structures (i.e., pili or adhesins). Bacterial genomes may encode multiple T4SSs, e.g., there are three functionally divergent T4SSs in some Bartonella species (vir, vbh, and trw). In a unique case, most rickettsial species encode a T4SS (rvh) enriched with gene duplication. Within single genomes, the evolutionary and functional implications of cross-system interchangeability of analogous T4SS protein components remains poorly understood. To lend insight into cross-system interchangeability, we analyzed the VirB8 family of T4SS channel proteins. Crystal structures of three VirB8 and two TrwG Bartonella proteins revealed highly conserved C-terminal periplasmic domain folds and dimerization interfaces, despite tremendous sequence divergence. This implies remarkable structural constraints for VirB8 components in the assembly of a functional T4SS. VirB8/TrwG heterodimers, determined via bacterial two-hybrid assays and molecular modeling, indicate that differential expression of trw and vir systems is the likely barrier to VirB8-TrwG interchangeability. We also determined the crystal structure of Rickettsia typhi RvhB8-II and modeled its coexpressed divergent paralog RvhB8-I. Remarkably, while RvhB8-I dimerizes and is structurally similar to other VirB8 proteins, the RvhB8-II dimer interface deviates substantially from other VirB8 structures, potentially preventing RvhB8-I/RvhB8-II heterodimerization. For the rvh T4SS, the evolution of divergent VirB8 paralogs implies a functional diversification that is unknown in other T4SSs. Collectively, our data identify two different constraints (spatiotemporal for Bartonella trw and vir T4SSs and structural for rvh T4SSs) that mediate the functionality of multiple divergent T4SSs within a single bacterium. IMPORTANCE:&nbsp; Assembly of multiprotein complexes at the right time and at the right cellular location is a fundamentally important task for any organism. In this respect, bacteria that express multiple analogous type IV secretion systems (T4SSs), each composed of around 12 different components, face an overwhelming complexity. Our work here presents the first structural investigation on factors regulating the maintenance of multiple T4SSs within a single bacterium. The structural data imply that the T4SS-expressing bacteria rely on two strategies to prevent cross-system interchangeability: (i) tight temporal regulation of expression or (ii) rapid diversification of the T4SS components. T4SSs are ideal drug targets provided that no analogous counterparts are known from eukaryotes. Drugs targeting the barriers to cross-system interchangeability (i.e., regulators) could dysregulate the structural and functional independence of discrete systems, potentially creating interference that prevents their efficient coordination throughout bacterial infection.</p

Evaluation of Two Models for Human Topoisomerase I Interaction with dsDNA and Camptothecin Derivatives

Human topoisomerase I (Top1) relaxes supercoiled DNA during cell division. Camptothecin stabilizes Top1/dsDNA covalent complexes which ultimately results in cell death, and this makes Top1 an anti-cancer target. There are two current models for how camptothecin and derivatives bind to Top1/dsDNA covalent complexes (Staker, et al., 2002, Proc Natl Acad Sci USA 99: 15387–15392; and Laco, et al., 2004, Bioorg Med Chem 12: 5225–5235). The interaction energies between bound camptothecin, and derivatives, and Top1/dsDNA in the two models were calculated. The published structure-activity-relationships for camptothecin and derivatives correlated with the interaction energies for camptothecin and derivatives in the Laco et al. model, however, this was not the case for several camptothecin derivatives in the Stacker et al. model. By defining the binding orientation of camptothecin and derivatives in the Top1/dsDNA active-site these results allow for the rational design of potentially more efficacious camptothecin derivatives

Biological and Structural Characterization of a Host-Adapting Amino Acid in Influenza Virus

Two amino acids (lysine at position 627 or asparagine at position 701) in the polymerase subunit PB2 protein are considered critical for the adaptation of avian influenza A viruses to mammals. However, the recently emerged pandemic H1N1 viruses lack these amino acids. Here, we report that a basic amino acid at position 591 of PB2 can compensate for the lack of lysine at position 627 and confers efficient viral replication to pandemic H1N1 viruses in mammals. Moreover, a basic amino acid at position 591 of PB2 substantially increased the lethality of an avian H5N1 virus in mice. We also present the X-ray crystallographic structure of the C-terminus of a pandemic H1N1 virus PB2 protein. Arginine at position 591 fills the cleft found in H5N1 PB2 proteins in this area, resulting in differences in surface shape and charge for H1N1 PB2 proteins. These differences may affect the protein's interaction with viral and/or cellular factors, and hence its ability to support virus replication in mammals

Leveraging structure determination with fragment screening for infectious disease drug targets: MECP synthase from Burkholderia pseudomallei

As part of the Seattle Structural Genomics Center for Infectious Disease, we seek to enhance structural genomics with ligand-bound structure data which can serve as a blueprint for structure-based drug design. We have adapted fragment-based screening methods to our structural genomics pipeline to generate multiple ligand-bound structures of high priority drug targets from pathogenic organisms. In this study, we report fragment screening methods and structure determination results for 2C-methyl-D-erythritol-2,4-cyclo-diphosphate (MECP) synthase from Burkholderia pseudomallei, the gram-negative bacterium which causes melioidosis. Screening by nuclear magnetic resonance spectroscopy as well as crystal soaking followed by X-ray diffraction led to the identification of several small molecules which bind this enzyme in a critical metabolic pathway. A series of complex structures obtained with screening hits reveal distinct binding pockets and a range of small molecules which form complexes with the target. Additional soaks with these compounds further demonstrate a subset of fragments to only bind the protein when present in specific combinations. This ensemble of fragment-bound complexes illuminates several characteristics of MECP synthase, including a previously unknown binding surface external to the catalytic active site. These ligand-bound structures now serve to guide medicinal chemists and structural biologists in rational design of novel inhibitors for this enzyme

SAD phasing using iodide ions in a high-throughput structural genomics environment

The Seattle Structural Genomics Center for Infectious Disease (SSGCID) focuses on the structure elucidation of potential drug targets from class A, B, and C infectious disease organisms. Many SSGCID targets are selected because they have homologs in other organisms that are validated drug targets with known structures. Thus, many SSGCID targets are expected to be solved by molecular replacement (MR), and reflective of this, all proteins are expressed in native form. However, many community request targets do not have homologs with known structures and not all internally selected targets readily solve by MR, necessitating experimental phase determination. We have adopted the use of iodide ion soaks and single wavelength anomalous dispersion (SAD) experiments as our primary method for de novo phasing. This method uses existing native crystals and in house data collection, resulting in rapid, low cost structure determination. Iodide ions are non-toxic and soluble at molar concentrations, facilitating binding at numerous hydrophobic or positively charged sites. We have used this technique across a wide range of crystallization conditions with successful structure determination in 16 of 17 cases within the first year of use (94% success rate). Here we present a general overview of this method as well as several examples including SAD phasing of proteins with novel folds and the combined use of SAD and MR for targets with weak MR solutions. These cases highlight the straightforward and powerful method of iodide ion SAD phasing in a high-throughput structural genomics environment

Classifying RNA-Binding Proteins Based on Electrostatic Properties

Protein structure can provide new insight into the biological function of a protein and can enable the design of better experiments to learn its biological roles. Moreover, deciphering the interactions of a protein with other molecules can contribute to the understanding of the protein's function within cellular processes. In this study, we apply a machine learning approach for classifying RNA-binding proteins based on their three-dimensional structures. The method is based on characterizing unique properties of electrostatic patches on the protein surface. Using an ensemble of general protein features and specific properties extracted from the electrostatic patches, we have trained a support vector machine (SVM) to distinguish RNA-binding proteins from other positively charged proteins that do not bind nucleic acids. Specifically, the method was applied on proteins possessing the RNA recognition motif (RRM) and successfully classified RNA-binding proteins from RRM domains involved in protein–protein interactions. Overall the method achieves 88% accuracy in classifying RNA-binding proteins, yet it cannot distinguish RNA from DNA binding proteins. Nevertheless, by applying a multiclass SVM approach we were able to classify the RNA-binding proteins based on their RNA targets, specifically, whether they bind a ribosomal RNA (rRNA), a transfer RNA (tRNA), or messenger RNA (mRNA). Finally, we present here an innovative approach that does not rely on sequence or structural homology and could be applied to identify novel RNA-binding proteins with unique folds and/or binding motifs

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

Structural Insight into How Bacteria Prevent Interference between Multiple Divergent Type IV Secretion Systems

Prokaryotes use type IV secretion systems (T4SSs) to translocate substrates (e.g., nucleoprotein, DNA, and protein) and/or elaborate surface structures (i.e., pili or adhesins). Bacterial genomes may encode multiple T4SSs, e.g., there are three functionally divergent T4SSs in some Bartonella species (vir, vbh, and trw). In a unique case, most rickettsial species encode a T4SS (rvh) enriched with gene duplication. Within single genomes, the evolutionary and functional implications of cross-system interchangeability of analogous T4SS protein components remains poorly understood. To lend insight into cross-system interchangeability, we analyzed the VirB8 family of T4SS channel proteins. Crystal structures of three VirB8 and two TrwG Bartonella proteins revealed highly conserved C-terminal periplasmic domain folds and dimerization interfaces, despite tremendous sequence divergence. This implies remarkable structural constraints for VirB8 components in the assembly of a functional T4SS. VirB8/TrwG heterodimers, determined via bacterial two-hybrid assays and molecular modeling, indicate that differential expression of trw and vir systems is the likely barrier to VirB8-TrwG interchangeability. We also determined the crystal structure of Rickettsia typhi RvhB8-II and modeled its coexpressed divergent paralog RvhB8-I. Remarkably, while RvhB8-I dimerizes and is structurally similar to other VirB8 proteins, the RvhB8-II dimer interface deviates substantially from other VirB8 structures, potentially preventing RvhB8-I/RvhB8-II heterodimerization. For the rvh T4SS, the evolution of divergent VirB8 paralogs implies a functional diversification that is unknown in other T4SSs. Collectively, our data identify two different constraints (spatio-temporal for Bartonella trw and vir T4SSs and structural for rvh T4SSs) that mediate the functionality of multiple divergent T4SSs within a single bacterium. IMPORTANCE Assembly of multiprotein complexes at the right time and at the right cellular location is a fundamentally important task for any organism. In this respect, bacteria that express multiple analogous type IV secretion systems (T4SSs), each composed of around 12 different components, face an overwhelming complexity. Our work here presents the first structural investigation on factors regulating the maintenance of multiple T4SSs within a single bacterium. The structural data imply that the T4SS-expressing bacteria rely on two strategies to prevent cross-system interchangeability: (i) tight temporal regulation of expression or (ii) rapid diversification of the T4SS components. T4SSs are ideal drug targets provided that no analogous counterparts are known from eukaryotes. Drugs targeting the barriers to cross-system interchangeability (i.e., regulators) could dysregulate the structural and functional independence of discrete systems, potentially creating interference that prevents their efficient coordination throughout bacterial infection.Peer reviewe