3D-Structural Homology Detection via Unassigned Residual Dipolar Couplings

Recognition of a protein\u27s fold provides valuable information about its function. While many sequence-based homology prediction methods exist, an important challenge remains: two highly dissimilar sequences can have similar folds --- how can we detect this rapidly, in the context of structural genomics? High-throughput NMR experiments, coupled with novel algorithms for data analysis, can address this challenge. We report an automated procedure for detecting 3D-structural homologies from sparse, unassigned protein NMR data. Our method identifies the 3D-structural models in a protein structural database whose geometries best fit the unassigned experimental NMR data. It does not use sequence information and is thus not limited by sequence homology. The method can also be used to confirm or refute structural predictions made by other techniques such as protein threading or sequence homology. The algorithm runs in O(pnk3) time, where p is the number of proteins in the database, n is the number of residues in the target protein, and k is the resolution of a rotation search. The method requires only uniform 15N-labelling of the protein and processes unassigned 1H-15N residual dipolar couplings, which can be acquired in a couple of hours. Our experiments on NMR data from 5 different proteins demonstrate that the method identifies closely related protein folds, despite low-sequence homology between the target protein and the computed model

High-Throughput 3D Homology Detection via NMR Resonance Assignment

One goal of the structural genomics initiative is the identification of new protein folds. Sequence-based structural homology prediction methods are an important means for prioritizing unknown proteins for structure determination. However, an important challenge remains: two highly dissimilar sequences can have similar folds --- how can we detect this rapidly, in the context of structural genomics? High-throughput NMR experiments, coupled with novel algorithms for data analysis, can address this challenge. We report an automated procedure, called HD, for detecting 3D structural homologies from sparse, unassigned protein NMR data. Our method identifies 3D models in a protein structural database whose geometries best fit the unassigned experimental NMR data. HD does not use, and is thus not limited by sequence homology. The method can also be used to confirm or refute structural predictions made by other techniques such as protein threading or homology modelling. The algorithm runs in $O(pn^{5/2} \log {(cn)} + p \log p)$ time, where $p$ is the number of proteins in the database, $n$ is the number of residues in the target protein and $c$ is the maximum edge weight in an integer-weighted bipartite graph. Our experiments on real NMR data from 3 different proteins against a database of 4,500 representative folds demonstrate that the method identifies closely related protein folds, including sub-domains of larger proteins, with as little as 10-30\% sequence homology between the target protein (or sub-domain) and the computed model. In particular, we report no false-negatives or false-positives despite significant percentages of missing experimental data

Structural characterization of intrinsically disordered proteins by NMR spectroscopy.

Recent advances in NMR methodology and techniques allow the structural investigation of biomolecules of increasing size with atomic resolution. NMR spectroscopy is especially well-suited for the study of intrinsically disordered proteins (IDPs) and intrinsically disordered regions (IDRs) which are in general highly flexible and do not have a well-defined secondary or tertiary structure under functional conditions. In the last decade, the important role of IDPs in many essential cellular processes has become more evident as the lack of a stable tertiary structure of many protagonists in signal transduction, transcription regulation and cell-cycle regulation has been discovered. The growing demand for structural data of IDPs required the development and adaption of methods such as 13C-direct detected experiments, paramagnetic relaxation enhancements (PREs) or residual dipolar couplings (RDCs) for the study of 'unstructured' molecules in vitro and in-cell. The information obtained by NMR can be processed with novel computational tools to generate conformational ensembles that visualize the conformations IDPs sample under functional conditions. Here, we address NMR experiments and strategies that enable the generation of detailed structural models of IDPs

Structural basis for sequence specific DNA binding and protein dimerization of HOXA13.

The homeobox gene (HOXA13) codes for a transcription factor protein that binds to AT-rich DNA sequences and controls expression of genes during embryonic morphogenesis. Here we present the NMR structure of HOXA13 homeodomain (A13DBD) bound to an 11-mer DNA duplex. A13DBD forms a dimer that binds to DNA with a dissociation constant of 7.5 nM. The A13DBD/DNA complex has a molar mass of 35 kDa consistent with two molecules of DNA bound at both ends of the A13DBD dimer. A13DBD contains an N-terminal arm (residues 324 - 329) that binds in the DNA minor groove, and a C-terminal helix (residues 362 - 382) that contacts the ATAA nucleotide sequence in the major groove. The N370 side-chain forms hydrogen bonds with the purine base of A5* (base paired with T5). Side-chain methyl groups of V373 form hydrophobic contacts with the pyrimidine methyl groups of T5, T6* and T7*, responsible for recognition of TAA in the DNA core. I366 makes similar methyl contacts with T3* and T4*. Mutants (I366A, N370A and V373G) all have decreased DNA binding and transcriptional activity. Exposed protein residues (R337, K343, and F344) make intermolecular contacts at the protein dimer interface. The mutation F344A weakens protein dimerization and lowers transcriptional activity by 76%. We conclude that the non-conserved residue, V373 is critical for structurally recognizing TAA in the major groove, and that HOXA13 dimerization is required to activate transcription of target genes

Structural characterization of intermolecular self-association in a T-cell specific kinase

This dissertation structurally examines the intermolecular self-association and quaternary structure of interleukin-2 tyrosine kinase (Itk) and draws conclusions about its relationship to the regulation and signaling of this immunologically important protein. Found primarily in haematopoietic cells, Itk is a member of the Tec family, the second largest family of non-receptor tyrosine kinases. All Tec family members share an SH3, SH2, and catalytic kinase domain structure. The activation of Itk occurs following T-cell receptor response to antigen stimulation. The exact mechanism of regulation in Tec family kinases is unclear. However, intermolecular self-association is emerging as a common characteristic among many members of the Tec family. To explore the potentially functionally significant self-association in Itk, high-resolution NMR solution structures were solved for the Itk SH3 domain, the Itk SH2 domain, and the Itk SH3/SH2 complex. The non-classical interaction between the Itk SH3 and SH2 domains mediates, in part, the self-association of full length Itk. The structure of the SH3/SH2 complex provides insight on how isomerization of a proline imide bond acts as an intrinsic molecular switch that preorganizes the CD loop of the SH2 domain for a non-classical interaction with the SH3 domain. Additionally, the oligomeric state of Itk self-association is characterized and the SH3/SH2 domain complex is used as a starting point to generate a structural model of the Itk SH3-SH2 fragment self-association that accounts for the oligomerization seen in native gel analysis. The same SH3/SH2 interaction is mutually exclusive with a quaternary structural rearrangement that supports autophosphorylation. Therefore, a structural model is described for Itk autophosphorylation that was generated using a previous point mutational analysis of the Itk SH2 domain coupled with covalent bond restraints found in the linkers of the SH3-SH2-kinase fragment. These studies bring us closer to understanding the structural mechanism behind Itk self-association and describe a model for one of many quaternary structural conformations in which Itk is likely to exist

Novel weak alignment techniques for nuclear magnetic resonance spectroscopy and applications to biomolecular structure determination

Nuclear magnetic resonance spectroscopy has continuously been developing ever since its introduction as a structural method in bioscience. Recently established residual dipolar coupling techniques yield information on long-range order in weakly aligned samples as they define the orientation of vectors between nuclei in a common global reference frame. These data complement classical short-range information and have a unique potential especially for the characterization of non-globular states. This thesis describes the development of novel methods for the weak alignment of biomacromolecules in charged gels and for the measurement of long-range residual dipolar couplings in perdeuterated proteins. These weak alignment techniques and other nuclear magnetic resonance methods were applied to study the structure and folding of various proteins such as the fibritin folding nucleus, the minicollagen cysteine rich domain and human protein tyrosine phosphatase 1B

Structural Elucidation and Functional Characterization of the Hyaloperonospora arabidopsidis Effector Protein ATR13

The oomycete Hyaloperonospora arabidopsidis (Hpa) is the causal agent of downy mildew on the model plant Arabidopsis thaliana and has been adapted as a model system to investigate pathogen virulence strategies and plant disease resistance mechanisms. Recognition of Hpa infection occurs when plant resistance proteins (R-genes) detect the presence or activity of pathogen-derived protein effectors delivered to the plant host. This study examines the Hpa effector ATR13 Emco5 and its recognition by RPP13-Nd, the cognate R-gene that triggers programmed cell death (HR) in the presence of recognized ATR13 variants. Herein, we use NMR to solve the backbone structure of ATR13 Emco5, revealing both a helical domain and a disordered internal loop. Additionally, we use site-directed and random mutagenesis to identify several amino acid residues involved in the recognition response conferred by RPP13-Nd. Using our structure as a scaffold, we map these residues to one of two surface-exposed patches of residues under diversifying selection. Exploring possible roles of the disordered region within the ATR13 structure, we perform domain swapping experiments and identify a peptide sequence involved in nucleolar localization. We conclude that ATR13 is a highly dynamic protein with no clear structural homologues that contains two surface-exposed patches of polymorphism, only one of which is involved in RPP13-Nd recognition specificity

Hybrid Approaches to Structural Characterization of Conformational Ensembles of Complex Macromolecular Systems Combining NMR Residual Dipolar Couplings and Solution X‑ray Scattering

Solving structures or structural ensembles of large macromolecular systems in solution poses a challenging problem. While NMR provides structural information at atomic resolution, increased spectral complexity, chemical shift overlap, and short transverse relaxation times (associated with slow tumbling) render application of the usual techniques that have been so successful for medium sized systems (\u3c50 \u3ekDa) difficult. Solution X-ray scattering, on the other hand, is not limited by molecular weight but only provides low resolution structural information related to the overall shape and size of the system under investigation. Here we review how combining atomic resolution structures of smaller domains with sparse experimental data afforded by NMR residual dipolar couplings (which yield both orientational and shape information) and solution X-ray scattering data in rigid-body simulated annealing calculations provides a powerful approach for investigating the structural aspects of conformational dynamics in large multidomain proteins. The application of this hybrid methodology is illustrated for the 128 kDa dimer of bacterial Enzyme I which exists in a variety of open and closed states that are sampled at various points in the catalytic cycles, and for the capsid protein of the human immunodeficiency virus