Structural Basis for the Interaction between the Potato Virus X Resistance Protein (Rx) and Its Cofactor Ran GTPase-activating Protein 2 (RanGAP2)

The potato (Solanum tuberosum) disease resistance protein Rx has a modular arrangement that contains coiled-coil (CC), nucleotide-binding (NB), and leucine-rich repeat (LRR) domains and mediates resistance to potato virus X. The Rx N-terminal CC domain undergoes an intramolecular interaction with the Rx NB-LRR region and an intermolecular interaction with the Rx cofactor RanGAP2 (Ran GTPase-activating protein 2). Here, we report the crystal structure of the Rx CC domain in complex with the Trp-Pro-Pro (WPP) domain of RanGAP2. The structure reveals that the Rx CC domain forms a heterodimer with RanGAP2, in striking contrast to the homodimeric structure of the CC domain of the barley disease resistance protein MLA10. Structure-based mutagenesis identified residues from both the Rx CC domain and the RanGAP2 WPP domain that are crucial for their interaction and function in vitro and in vivo. Our results reveal the molecular mechanism underlying the interaction of Rx with RanGAP2 and identify the distinct surfaces of the Rx CC domain that are involved in intramolecular and intermolecular interactions.     

Folded-unfolded cross-predictions and protein evolution: The case study of coiled-coils

Here we report a thorough analysis of cross-predictions between coiled-coil and disordered protein segments using various prediction algorithms for both sequence classes. Coiled-coils are often predicted to be unstructured, consistent with their obligate multimeric nature, whereas reverse cross-predictions are rare due to the regularity of coiled-coil sequences. We propose the simultaneous use of the programs Coils and IUPred to achieve acceptable prediction accuracy and minimize the extent of cross-predictions. The relevance of observed cross-predictions might be that disordered sequences can adopt coiled-coil conformation relatively easily during protein evolution.     

Characterization of Rab45/RASEF containing EF-hand domain and a coiled-coil motif as a self-associating GTPase

Rab-family GTPases function as key regulators for membrane traffic. Among them, Rab45/RASEF is an atypical GTPase in that it contains a coiled-coil motif at the mid region and a distinct N-terminal EF-hand domain with C-terminal Rab-homology domain. Here, we provide the initial biochemical characterization and intracellular localization of human Rab45. Rab45 bound guanine nucleotide tri- and di-phosphates through the C-terminal Rab domain. Rab45 was capable of self-interacting, and the self-interaction required the mid region containing the coiled-coil motif. Rab45 expressed in HeLa cells was localized in a small patch in the perinuclear area of the cell, and the localization was regulated by the guanine nucleotide-bound states of Rab45. Interestingly, the mid region, together with Rab domain, appeared to be essential for the characteristic perinuclear localization of Rab45, indicating that the self-interaction may be involved in the intracellular localization of Rab45.     

Genetic variation in the flanking regions of Shiga toxin 2 gene in Shiga toxin-producing Escherichia coli O157:H7 isolated in Japan

PM61 is a chain-forming envC strain of Escherichia coli with a leaky outer membrane. It was found to have an oversized penicillin-binding protein 3, which was the result of an IS4 insertion in the prc gene. The other properties of PM61 were caused by the envC mutation. We cloned the envC (yibP) gene and identified the mutation site, causing a single residue substitution, H366Y, in the PM61 envC allele. The gene product was predicted to be a periplasmic protein having coiled-coil structure in the N-terminal region and homology to lysostaphin in the C-terminal region. Overexpression of envC inhibited cell growth, and overexpression of the PM61 mutant allele caused cell lysis. Disruption of the chromosomal envC caused the same defects as the envC point mutation, indicating the gene is dispensable for growth but important for normal septation/separation and cell envelope integrity.     

Identification and characterization of novel NuMA isoforms

The large nuclear mitotic apparatus (NuMA) has been investigated for over 30 years with functions related to the formation and maintenance of mitotic spindle poles during mitosis. However, the existence and functions of NuMA isoforms generated by alternative splicing remains unclear. In the present work, we show that at least seven NuMA isoforms (categorized into long, middle and short groups) generated by alternative splicing from a common NuMA mRNA precursor were discovered in HeLa cells and these isoforms differ mainly at the carboxyl terminus and the coiled-coil domains. Two “hotspot” exons with molecular mass of 3366-nt and 42-nt tend to be spliced during alternative splicing in long and middle groups. Furthermore, full-length coding sequences of long and middle NuMA obtained by using fusion PCR were constructed into GFP-tagged vector to illustrate their cellular localization. Long NuMA mainly localized in the nucleus with absence from nucleoli during interphase and translocated to the spindle poles in mitosis. Middle NuMA displayed the similar cell cycle-dependent distribution pattern as long NuMA. However, expression of NuMA short isoforms revealed a distinct subcellular localization. Short NuMA were present in the cytosol during the whole cycle, without colocalization with mitotic apparatus. These results have allowed us tentatively to explore a new research direction for NuMA’s various functions.     

Isolation and mapping of a putative b subunit of human ATP synthase (ATP-BL) from human leukocytes.

From a human-leukocyte cDNA library, we cloned cDNA encoding a novel protein, which has a significant homology with the b subunit of ATP synthase (proton-transporting ATPase, F1F0-ATPase; EC3.6.1.34) derived from Anabaena sp. strain PCC 7120. The cDNA has an open reading frame of 1314 nucleotides corresponding to 438 amino acids. The coding sequence was 37.9% identical over 57 amino acid with b subunit of ATP synthase. The 34-amino-acid region of the predicted peptide sequence displays a coiled-coil motif that could form a complex with some other protein(s). We designated this novel gene as ATP-BL because of its homology to the b subunit of ATP synthase. The ATP-BL locus was mapped by fluorescence in situ hybridization (FISH) and radiation hybrid mapping to the q24 region of chromosome 16.     

Structure and flexibility of the tropomyosin overlap junction

To be effective as a gatekeeper regulating the access of binding proteins to the actin filament, adjacent tropomyosin molecules associate head-to-tail to form a continuous super-helical cable running along the filament surface. Chimeric head-to-tail structures have been solved by NMR and X-ray crystallography for N- and C-terminal segments of smooth and striated muscle tropomyosin spliced onto non-native coiled-coil forming peptides. The resulting 4-helix complexes have a tight coiled-coil N-terminus inserted into a separated pair of C-terminal helices, with some helical unfolding of the terminal chains in the striated muscle peptides. These overlap complexes are distinctly curved, much more so than elsewhere along the superhelical tropomyosin cable. To verify whether the non-native protein adducts (needed to stabilize the coiled-coil chimeras) perturb the overlap, we carried out Molecular Dynamics simulations of head-to-tail structures having only native tropomyosin sequences. We observe that the splayed chains all refold and become helical. Significantly, the curvature of both the smooth and the striated muscle overlap domain is reduced and becomes comparable to that of the rest of the tropomyosin cable. Moreover, the measured flexibility across the junction is small. This and the reduced curvature ensure that the super-helical cable matches the contours of F-actin without manifesting localized kinking and excessive flexibility, thus enabling the high degree of cooperativity in the regulation of myosin accessibility to actin filaments.