Intramolecular autoregulatory sequences in proteins

The worldwide structural genomics initiative promises, within the next decade, to provide us with the three-dimensional structures of most representative proteins. However, the initiative does not in the initial stages specifically address the structural basis of protein-protein interactions and protein regulation, both of which are crucial for understanding the cellular function of proteins. This report reviews the studies of the structural basis of protein-protein interactions and regulation in several biologically important cellular processes carried out in our laboratory. The focus will be on the recognition of nuclear localization sequences (NLSs) by the nuclear import factor importin-alpha, and its regulation. Regulatory interactions in retroviral envelope proteins and the enzyme phenylalanine hydroxylase (PAH) will be discussed also. All three biological systems share a regulatory mechanism termed active site-directed or intrasteric regulation

Protein regulation, protein-protein interactions and structural genomics

The new technical developments and the success of genome sequencing projects have prompted a new approach to scientific investigation and discovery in every field of biochemistry and molecular biology, including structural biology. One of the most prominent recent developments is the birth of structural genomics, a world-wide initiative that aims to provide the three-dimensional structures of all representative proteins. However, structural biology faces an exciting future beyond structural genomics; if we are to understand how the proteome works and use the genomic information for therapeutic purposes, studies of protein-protein interactions and macromolecular complexes, mechanism and regulation of macromolecular function, membrane protein structure, and structure-based therapeutic design must be pursued in parallel. Successful approaches will combine large-scale, high-throughput approaches developed through structural genomics with more traditional hypothesis-driven approaches, supported by integrative bioinformatics tools

United we stand: combining structural methods

Structural biologists benefit enormously by combining structural approaches to tackle biological systems. This is evident in the increasing use of complementary methods combined with the traditional structural biology techniques of macromolecular X-ray crystallography (MX), nuclear magnetic resonance (NMR) and electron microscopy (EM) to generate structural information

Factors influencing nucleo-cytoplasmic trafficking: which matter? Response to Alvisi & Jans' comment on Phosphorylation adjacent to the nuclear localization signal of human dUTPase abolishes nuclear import: structural and mechanistic insights

The authors respond to a comment by Alvisi & Jans [(2014), Acta Cryst. D70, 2775-2776] on the article Phosphorylation adjacent to the nuclear localization signal of human dUTPase abolishes nuclear import: structural and mechanistic insights [Róna et al. (2013), Acta Cryst. D69, 2495-2505]

Heterogeneous nucleation is required for crystallization of the ZnuA domain of pneumococcal AdcA

Zn2+ is an essential nutrient for all known forms of life. In the major human pathogen Streptococcus pneumoniae, the acquisition of Zn2+ is facilitated by two Zn2+-specific solute-binding proteins: AdcA and AdcAII. To date, there has been a paucity of structural information on AdcA, which has hindered a deeper understanding of the mechanism underlying pneumococcal Zn2+ acquisition. Native AdcA consists of two domains: an N-terminal ZnuA domain and a C-terminal ZinT domain. In this study, the ZnuA domain of AdcA was crystallized. The initial crystals of the ZnuA-domain protein were obtained using dried seaweed as a heterogeneous nucleating agent. No crystals were obtained in the absence of the heterogeneous nucleating agent. These initial crystals were subsequently used as seeds to produce diffraction-quality crystals. The crystals diffracted to 2.03 angstrom resolution and had the symmetry of space group P1. This study demonstrates the utility of heterogeneous nucleation. The solution of the crystal structures will lead to further understanding of Zn2+ acquisition by S. pneumoniae

Fido, a Novel AMPylation Domain Common to Fic, Doc, and AvrB

BACKGROUND:The Vibrio parahaemolyticus type III secreted effector VopS contains a fic domain that covalently modifies Rho GTPase threonine with AMP to inhibit downstream signaling events in host cells. The VopS fic domain includes a conserved sequence motif (HPFx[D/E]GN[G/K]R) that contributes to AMPylation. Fic domains are found in a variety of species, including bacteria, a few archaea, and metazoan eukaryotes. METHODOLOGY/PRINCIPAL FINDINGS:We show that the AMPylation activity extends to a eukaryotic fic domain in Drosophila melanogaster CG9523, and use sequence and structure based computational methods to identify related domains in doc toxins and the type III effector AvrB. The conserved sequence motif that contributes to AMPylation unites fic with doc. Although AvrB lacks this motif, its structure reveals a similar topology to the fic and doc folds. AvrB binds to a peptide fragment of its host virulence target in a similar manner as fic binds peptide substrate. AvrB also orients a phosphate group from a bound ADP ligand near the peptide-binding site and in a similar position as a bound fic phosphate. CONCLUSIONS/SIGNIFICANCE:The demonstrated eukaryotic fic domain AMPylation activity suggests that the VopS effector has exploited a novel host posttranslational modification. Fic domain-related structures give insight to the AMPylation active site and to the VopS fic domain interaction with its host GTPase target. These results suggest that fic, doc, and AvrB stem from a common ancestor that has evolved to AMPylate protein substrates

Crystallization, X-ray diffraction analysis and preliminary structure determination of the TIR domain from the flax resistance protein L6

The Toll/interleukin-1 receptor (TIR) domain is a protein-protein interaction domain that is found in both animal and plant immune receptors. In animal Toll-like receptor signalling, both homotypic TIR-domain interactions between two receptor molecules and heterotypic interactions between receptors and TIR-domain-containing adaptors are required for initiation of an innate immune response. The TIR domains in cytoplasmic nucleotide-binding/leucine-rich repeat (NB-LRR) plant disease-resistance proteins are not as well characterized, but recent studies have suggested a role in defence signalling. In this study, the crystallization, X-ray diffraction analysis and preliminary structure determination of the TIR domain from the flax resistance protein L6 (L6TIR) are reported. Plate-like crystals of L6TIR were obtained using PEG 200 as a precipitant and diffracted X-rays to 2.3 angstrom resolution. Pseudo-translation complicated the initial assignment of the crystal symmetry, which was ultimately found to correspond to space group P2(1)2(1)2 with two molecules per asymmetric unit. The structure of L6TIR was solved by molecular replacement using the structure of the TIR-domain-containing protein AT1G72930 from Arabidopsis as a template

Towards the structure of the TIR-domain signalosome

TIR (Toll/interleukin-1 receptor/resistance protein) domains feature in animal, plant and bacterial proteins involved in innate immunity pathways and associated processes. They function through protein:protein interactions, in particular self-association and homotypic association with other TIR domains. Structures of TIR domains from all phyla have been determined, but common association modes have only emerged for plant and bacterial TIR domains, and not for mammalian TIR domains. Numerous attempts involving hybrid approaches, which have combined structural, computational, mutagenesis and biophysical data, have failed to converge onto common models of how these domains associate and function. We propose that the available data can be reconciled in the context of higher-order assembly formation, and that TIR domains function through signaling by cooperative assembly formation (SCAF).The work in the authors’ laboratories was supported by the National Health and Medical Research Council (NHMRC grants 1003326, 1107804, 1071659) and the Australian Research Council (ARC Discovery Projects DP120100685, DP160102244). BK is NHMRC Principal Research Fellow (1003325, 1110971). Simon Williams is funded by ARC DECRA (DE160100893). We acknowledge the use of the University of Queensland Remote Operation Crystallization and X-ray Diffraction Facility (UQ ROCX) and the Australian Synchrotron (MX and SAXSWAXS beamlines) for our structural work