Scott A. McCallum

Structure of C3b in complex with CRIg gives insights into regulation of complement activation.

The complement system is a key part of the innate immune system, and is required for clearance of pathogens from the bloodstream. After exposure to pathogens, the third component of the complement system, C3, is cleaved to C3b which, after recruitment of factor B, initiates formation of the alternative pathway convertases. CRIg, a complement receptor expressed on macrophages, binds to C3b and iC3b mediating phagocytosis of the particles, but it is unknown how CRIg selectively recognizes proteolytic C3-fragments and whether binding of CRIg to C3b inhibits convertase activation. Here we present the crystal structure of C3b in complex with CRIg and, using CRIg mutants, provide evidence that CRIg acts as an inhibitor of the alternative pathway of complement. The structure shows that activation of C3 induces major structural rearrangements, including a dramatic movement (>80u2009Å) of the thioester-bond-containing domain through which C3b attaches to pathogen surfaces. We show that CRIg is not only a phagocytic receptor, but also a potent inhibitor of the alternative pathway convertases. The structure provides insights into the complex macromolecular structural rearrangements that occur during complement activation and inhibition. Moreover, our structure–function studies relating the structural basis of complement activation and the means by which CRIg inhibits the convertases provide important clues to the development of therapeutics that target complement.

NMR Methods for Studying the Structure and Dynamics of RNA

Proper functioning of RNAs requires the formation of complex three‐dimensional structures combined with the ability to rapidly interconvert between multiple functional states. This review covers recent advances in isotope‐labeling strategies and NMR experimental approaches that have promise for facilitating solution structure determinations and dynamics studies of biologically active RNAs. Improved methods for the production of isotopically labeled RNAs combined with new multidimensional heteronuclear NMR experiments make it possible to dramatically reduce spectral crowding and simplify resonance assignments for RNAs. Several novel applications of experiments that directly detect hydrogen‐bonding interactions are discussed. These studies demonstrate how NMR spectroscopy can be used to distinguish between possible secondary structures and identify mechanisms of ligand binding in RNAs. A variety of recently developed methods for measuring base and sugar residual dipolar couplings are described. NMR residual dipolar coupling techniques provide valuable data for determining the long‐range structure and orientation of helical regions in RNAs. A number of studies are also presented where residual dipolar coupling constraints are used to determine the global structure and dynamics of RNAs. NMR relaxation data can be used to probe the dynamics of macromolecules in solution. The power dependence of transverse rotating‐frame relaxation rates was used here to study dynamics in the minimal hammerhead ribozyme. Improved methods for isotopically labeling RNAs combined with new types of structural data obtained from a growing repertoire of NMR experiments are facilitating structural and dynamic studies of larger RNAs.

MONTE: An automated Monte Carlo based approach to nuclear magnetic resonance assignment of proteins.

A general-purpose Monte Carlo assignment program has been developed to aid in the assignment of NMR resonances from proteins. By virtue of its flexible data requirements the program is capable of obtaining assignments of both heavily deuterated and fully protonated proteins. A wide variety of source data, such as inter-residue scalar connectivity, inter-residue dipolar (NOE) connectivity, and residue specific information, can be utilized in the assignment process. The program can also use known assignments from one form of a protein to facilitate the assignment of another form of the protein. This attribute is useful for assigning protein-ligand complexes when the assignments of the unliganded protein are known. The program can be also be used as an interactive research tool to assist in the choice of additional experimental data to facilitate completion of assignments. The assignment of a deuterated 45xa0kDa homodimeric Glutathione-S-transferase illustrates the principal features of the program.

Refined Solution Structure of the Iron-responsive Element RNA Using Residual Dipolar Couplings

The iron-responsive element (IRE) is a 30nt RNA motif located in the non-coding regions of mRNAs of proteins involved in iron regulation. In humans, the IRE plays a direct role in the control of iron levels by post-transcriptional regulation of the ferritin and transferrin receptor proteins through highly specific recognition by IRE-binding proteins. The IRE fold is representative of many RNA motifs that contain helical domains separated by a bulge or internal loop. The global structures of such extended multi-domain RNAs are not well defined by conventional NMR-distance and torsion angle structural restraints. Residual dipolar couplings (RDCs) are employed here to better define the global structure of the IRE RNA in solution. RDCs contain valuable long-range structural information that compliments the short-range structural data derived from standard NOE-distance and torsion angle restraints. Several approaches for estimating alignment tensor parameters and incorporating RDCs into RNA structure determinations are compared. Both the local and global structure of the IRE are improved significantly by refinement with RDCs. These RDC refinements provide insight on the conformational dynamics of the IRE. These studies highlight some issues that need to be addressed when incorporating RDCs in solution structure determinations of nucleic acids. The approach used here should prove valuable for structure determinations of various multi-domain systems.

Role of a heterogeneous free state in the formation of a specific RNA-theophylline complex.

The helical regions of RNA are generally very stable, but the single-stranded and loop regions often exist as an ensemble of conformations in solution. The theophylline-binding RNA aptamer forms a very stable structure when bound to the bronchodilator theophylline, but the theophylline binding site is not stably formed in the absence of ligand. The kinetics for theophylline binding were measured here by stopped-flow fluorescence spectroscopy to probe the mechanism for theophylline binding in this RNA aptamer. The kinetic studies showed that formation of the RNA-theophylline complex is over 1000 times slower than a diffusion-controlled rate, and the high affinity of the RNA-theophylline complex arises primarily from a slow dissociation rate for the complex. A theophylline-independent rate was observed for formation of the theophylline-RNA complex at high theophylline concentration, indicating that a conformational change in the RNA is the rate-limiting step in complex formation under these conditions. The RNA-theophylline complex requires divalent metal ions, such as Mg2+, to form a high-affinity complex, and there is a greater than 10000-fold reduction in affinity for theophylline in the absence of Mg2+. This decrease in binding affinity in the absence of Mg2+ results primarily from an increased dissociation rate for the complex. The implications of an ensemble of conformations in the free state of this theophylline-binding RNA are discussed and compared with mechanisms for formation of protein-ligand complexes.

Data requirements for reliable chemical shift assignments in deuterated proteins

The information required for chemical shift assignments in large deuterated proteins was investigated using a Monte Carlo approach (Hitchens etxa0al., 2002). In particular, the consequences of missing amide resonances on the reliability of assignments derived from Cα and CO or from Cα and Cβ chemical shifts was investigated. Missing amide resonances reduce both the number of correct assignments as well as the confidence in these assignments. More significantly, a number of undetectable errors can arise when as few as 9% of the amide resonances are missing from the spectra. However, the use of information from residue specific labeling as well as local and long-range distance constraints improves the reliability and extent of assignment. It is also shown that missing residues have only a minor effect on the assignment of protein-ligand complexes using Cα and CO chemical shifts and Cα inter-residue connectivity, provided that the known chemical shifts of the unliganded protein are utilized in the assignment process.