A novel BICD2 mutation of a patient with Spinal Muscular Atrophy Lower Extremity Predominant 2.

The bicaudal D homolog 2 (BICD2) gene encodes a protein required for the stable complex of dynein and dynactin, which functions as a motor protein working along the microtubule cytoskeleton. Both inherited and de novo variants of BICD2 are reported with autosomal dominant spinal muscular atrophy with lower extremity predominance (SMALED2). Here, we report a male patient with a novel mutation in the BICD2 gene caused by a heterozygous substitution of arginine with cysteine at residue 162 (Arg162Cys); inherited from his asymptomatic mother. The patient showed typical clinical symptoms of SMALED2, which was genetically confirmed by sequencing. The Arg162Cys mutant clusters with four previously reported variants (c.361C>G, p.Leu121Val; c.581A>G, p.Gln194Arg; c.320C>T, p.Ser107Leu; c.565A>T, p.Ile189Phe) in a region that binds to the dynein-dynactin complex (DDC). The BICD2 domain structures were predicted and the Arg162Cys mutation was localized in the N-terminus coiled-coil segment 1 (CC1) domain. Protein modeling of BICD2's CC1 domain predicted that the Arg162Cys missense variant disrupted interactions with dynein cytoplasmic 1 heavy chain 1 within the DDC. The mutant did this by either changing the electrostatic surface potential or making a broader hydrophobic unit with the neighboring residues. This hereditary case supports the complex and broad genotype-phenotype correlation of BICD2 mutations, which could be explained by incomplete penetrance or variable expressivity in the next generation.

Intramolecular interaction in LGN, an adaptor protein that regulates mitotic spindle orientation.

Proper mitotic spindle orientation requires that astral microtubules are connected to the cell cortex by the microtubule-binding protein NuMA, which is recruited from the cytoplasm. Cortical recruitment of NuMA is at least partially mediated via direct binding to the adaptor protein LGN. LGN normally adopts a closed conformation via an intramolecular interaction between its N-terminal NuMA-binding domain and its C-terminal region that contains four GoLoco (GL) motifs, each capable of binding to the membrane-anchored Gαi subunit of heterotrimeric G protein. Here we show that the intramolecular association with the N-terminal domain in LGN involves GL3, GL4, and a region between GL2 and GL3, whereas GL1 and GL2 do not play a major role. This conformation renders GL1 but not the other GL motifs in a state easily accessible to Gαi To interact with full-length LGN in a closed state, NuMA requires the presence of Gαi; both NuMA and Gαi are essential for cortical recruitment of LGN in mitotic cells. In contrast, mInsc, a protein that competes with NuMA for binding to LGN and regulates mitotic spindle orientation in asymmetric cell division, efficiently binds to full-length LGN without Gαi and induces its conformational change, enhancing its association with Gαi In nonpolarized symmetrically dividing HeLa cells, disruption of the LGN-NuMA interaction by ectopic expression of mInsc results in a loss of cortical localization of NuMA during metaphase and anaphase and promotes mitotic spindle misorientation and a delayed anaphase progression. These findings highlight a specific role for LGN-mediated cell cortex recruitment of NuMA.

Ric-8A-mediated stabilization of the trimeric G protein subunit Gαi is inhibited by pertussis toxin-catalyzed ADP-ribosylation.

The heterotrimeric G protein subunit Gαi can be activated by G protein-coupled receptors and the cytosolic protein Ric-8A, the latter of which is also known to prevent ubiquitin-dependent degradation of Gαi. Here we show that the amounts of the three Gαi-related proteins Gαi1, Gαi2, and Gαi3, but not that of Gαq, are rapidly decreased by cell treatment with pertussis toxin (PTX). The decrease appears to be due to ADP-ribosylation of Gαi, because PTX treatment does not affect the amount of a mutant Gαi2 carrying alanine substitution for Cys352, the residue that is ADP-ribosylated by the toxin. The presence of endogenous and exogenous Ric-8A increases Gαi stability as shown in cells treated with the protein synthesis inhibitor cycloheximide; however, Ric-8A fails to efficiently stabilize ADP-ribosylated Gαi. The failure agrees with the inability of Ric-8A to bind to ADP-ribosylated Gαi both in vitro and in vivo. Thus PTX appears to exert its pathological effects at least in part by converting Gαi to an unstable ADP-ribosylated form, in addition to the well-known inability of ADP-ribosylated Gαi to transduce signals triggered by G protein-coupled receptors.

Structural basis for the recognition of the scaffold protein Frmpd4/Preso1 by the TPR domain of the adaptor protein LGN.

The adaptor protein LGN interacts via the N-terminal domain comprising eight tetratricopeptide-repeat (TPR) motifs with its partner proteins mInsc, NuMA, Frmpd1 and Frmpd4 in a mutually exclusive manner. Here, the crystal structure of the LGN TPR domain in complex with human Frmpd4 is described at 1.5 Å resolution. In the complex, the LGN-binding region of Frmpd4 (amino-acid residues 990-1011) adopts an extended structure that runs antiparallel to LGN along the concave surface of the superhelix formed by the TPR motifs. Comparison with the previously determined structures of the LGN-Frmpd1, LGN-mInsc and LGN-NuMA complexes reveals that these partner proteins interact with LGN TPR1-6 via a common core binding region with consensus sequence (E/Q)XEX4-5(E/D/Q)X1-2(K/R)X0-1(V/I). In contrast to Frmpd1, Frmpd4 makes additional contacts with LGN via regions N- and C-terminal to the core sequence. The N-terminal extension is replaced by a specific α-helix in mInsc, which drastically increases the direct contacts with LGN TPR7/8, consistent with the higher affinity of mInsc for LGN. A crystal structure of Frmpd4-bound LGN in an oxidized form is also reported, although oxidation does not appear to strongly affect the interaction with Frmpd4.

Structural basis of cofactor-mediated stabilization and substrate recognition of the α-tubulin acetyltransferase αTAT1.

The functions of microtubules are controlled in part by tubulin post-translational modification including acetylation of Lys4° in α-tubulin. αTAT1 (α-tubulin acetyltransferase 1), an enzyme evolutionarily conserved among eukaryotes, has recently been identified as the major α-tubulin Lys4° acetyltransferase, in which AcCoA (acetyl-CoA) serves as an acetyl group donor. The regulation and substrate recognition of this enzyme, however, have not been fully understood. In the present study, we show that AcCoA and CoA each form a stable complex with human αTAT1 to maintain the protein integrity both in vivo and in vitro. The invariant residues Arg¹³² and Ser¹6° in αTAT1 participate in the stable interaction not only with AcCoA but also with CoA, which is supported by analysis of the present crystal structures of the αTAT1 catalytic domain in complex with CoA. Alanine substitution for Arg¹³² or Ser¹6° leads to a drastic misfolding of the isolated αTAT1 catalytic domain in the absence of CoA and AcCoA but not in the presence of excess amounts of either cofactor. A mutant αTAT1 carrying the R132A or S160A substitution is degraded much faster than the wild-type protein when expressed in mammalian Madin-Darby canine kidney cells. Furthermore, alanine-scanning experiments using Lys4°-containing peptides reveal that α-tubulin Ser³8 is crucial for substrate recognition of αTAT1, whereas Asp³9, Ile4², the glycine stretch (amino acid residues 43-45) and Asp46 are also involved. The requirement for substrate selection is totally different from that in various histone acetyltransferases, which appears to be consistent with the inability of αTAT1 to acetylate histones.

Ubiquitination of the heterotrimeric G protein α subunits Gαi2 and Gαq is prevented by the guanine nucleotide exchange factor Ric-8A.

The cytosolic protein Ric-8A acts as a guanine nucleotide exchange factor for Gα subunits of the Gi, Gq, and G12/13 classes of heterotrimeric G protein in vitro, and is also known to increase the amounts of these Gα proteins in vivo. The mechanism whereby Ric-8 regulates Gα content, however, has not been fully understood. Here we show that Ric-8 Astabilizes Gαi2 and Gαq by preventing their ubiquitination. Ric-8A interacts with and stabilizes Gαi2, Gαq, Gα12, but not Gαs, when expressed in COS-7 cells. The protein levels of Gαi2 and Gαq appear to be controlled via the ubiquitin-proteasome degradation pathway, because these Gα subunits undergo polyubiquitination and are stabilized with the proteasome inhibitor MG132. The ubiquitination of Gαi2 and Gαq is suppressed by expression of Ric-8A. The suppression likely requires Ric-8A interaction with these Gα proteins; the C-terminal truncation of Gαq and Gαi2 completely abrogates their interaction with Ric-8A, their stabilization by Ric-8A, and Ric-8A-mediated inhibition of Gα ubiquitination.

Crystal structure of the C-terminal globular domain of oligosaccharyltransferase from Archaeoglobus fulgidus at 1.75 Å resolution.

Protein N-glycosylation occurs in the three domains of life. Oligosaccharyltransferase (OST) transfers glycan to asparagine in the N-glycosylation sequon. The catalytic subunit of OST is called STT3 in eukaryotes, AglB in archaea, and PglB in eubacteria. The genome of a hyperthermophilic archaeon, Archaeoglobus fulgidus, encodes three AglB paralogs. Two of them are the shortest AglBs across all domains of life. We determined the crystal structure of the C-terminal globular domain of the smallest AglB to identify the minimal structural unit. The Archaeoglobus AglB lacked a ß-barrel-like structure, which had been found in other AglB and PglB structures. In agreement, the deletion in a larger Pyrococcus AglB confirmed its dispensability for the activity. By contrast, the Archaeoglobus AglB contains a kinked helix bearing a conserved motif, called DK/MI motif. The lysine and isoleucine residues in the motif participate in the Ser/Thr recognition in the sequon. The Archaeoglobus AglB structure revealed that the kinked helix contained an unexpected insertion. A revised sequence alignment based on this finding identified a variant type of the DK motif with the insertion. A mutagenesis study of the Archaeoglobus AglB confirmed the contribution of this particular type of the DK motif to the activity. When taken together with our previous results, this study defined the classification of OST: one group consisting of eukaryotes and most archaea possesses the DK-type Ser/Thr pocket, and the other group consisting of eubacteria and the remaining archaea possesses the MI-type Ser/Thr pocket. This classification provides a useful framework for OST studies.

Structural basis for interaction between the conserved cell polarity proteins Inscuteable and Leu-Gly-Asn repeat-enriched protein (LGN).

Interaction between the mammalian cell polarity proteins mInsc (mammalian homologue of Inscuteable) and Leu-Gly-Asn repeat-enriched protein (LGN), as well as that between their respective Drosophila homologues Inscuteable and Partner of Inscuteable (Pins), plays crucial roles in mitotic spindle orientation, a process contributing to asymmetric cell division. Here, we report a crystal structure of the LGN-binding domain (LBD) of human mInsc complexed with the N-terminal tetratricopeptide repeat (TPR) motifs of human LGN at 2.6-Å resolution. In the complex, mInsc-LBD adopts an elongated structure with three binding modules--an α-helix, an extended region, and a ß-sheet connected with a loop--that runs antiparallel to LGN along the concave surface of the superhelix formed by the TPRs. Structural analysis and structure-based mutagenesis define residues that are critical for mInsc-LGN association, and reveal that the activator of G-protein signaling 3 (AGS3)-binding protein Frmpd1 [4.1/ezrin/radixin/moesin (FERM) and PSD-95/Dlg/ZO-1 (PDZ) domain-containing protein 1] and its relative Frmpd4 interact with LGN via a region homologous to a part of mInsc-LBD, whereas nuclear mitotic apparatus protein (NuMA) and the C terminus of LGN recognize the TPR domain in a manner different from that by mInsc. mInsc binds to LGN with the highest affinity (K(D) ≈ 2.4 nM) and effectively replaces the Frmpd proteins, NuMA, and the LGN C terminus, suggesting the priority of mInsc in binding to LGN. We also demonstrate, using mutant proteins, that mInsc-LGN interaction is vital for stabilization of LGN and for intracellular localization of mInsc.

A conserved region between the TPR and activation domains of p67phox participates in activation of the phagocyte NADPH oxidase.

The phagocyte NADPH oxidase, dormant in resting cells, is activated during phagocytosis to produce superoxide, a precursor of microbicidal oxidants. The membrane-integrated protein gp91(phox) serves as the catalytic core, because it contains a complete electron-transporting apparatus from NADPH to molecular oxygen for superoxide production. Activation of gp91(phox) requires the cytosolic proteins p67(phox), p47(phox), and Rac (a small GTPase). p67(phox), comprising 526 amino acids, moves upon cell stimulation to the membrane together with p47(phox) and there interacts with Rac; these processes are prerequisite for gp91(phox) activation. Here we show that a region of p67(phox) (amino acids 190-200) C-terminal to the Rac-binding domain is evolutionarily well conserved and participates in oxidase activation at a later stage in conjunction with an activation domain. Alanine substitution for Tyr-198, Leu-199, or Val-204 abrogates the ability of p67(phox) to support superoxide production by gp91(phox)-based oxidase as well as its related oxidases Nox1 and Nox3; the activation also involves other invariant residues such as Leu-193, Asp-197, and Gly-200. Intriguingly, replacement of Gln-192 by alanine or that of Tyr-198 by phenylalanine or tryptophan rather enhances superoxide production by gp91(phox)-based oxidase, suggesting a tuning role for these residues. Furthermore, the Y198A/V204A or L199A/V204A substitution leads to not only a complete loss of the activity of the reconstituted oxidase system but also a significant decrease in p67(phox) interaction with the gp91(phox) NADPH-binding domain, although these mutations affect neither the protein integrity nor the Rac binding activity. Thus the extended activation domain of p67(phox) (amino acids 190-210) containing the D(Y/F)LGK motif plays an essential role in oxidase activation probably by interacting with gp91(phox).

The selectivity of receptor tyrosine kinase signaling is controlled by a secondary SH2 domain binding site.

SH2 domain-mediated interactions represent a crucial step in transmembrane signaling by receptor tyrosine kinases. SH2 domains recognize phosphotyrosine (pY) in the context of particular sequence motifs in receptor phosphorylation sites. However, the modest binding affinity of SH2 domains to pY containing peptides may not account for and likely represents an oversimplified mechanism for regulation of selectivity of signaling pathways in living cells. Here we describe the crystal structure of the activated tyrosine kinase domain of FGFR1 in complex with a phospholipase Cgamma fragment. The structural and biochemical data and experiments with cultured cells show that the selectivity of phospholipase Cgamma binding and signaling via activated FGFR1 are determined by interactions between a secondary binding site on an SH2 domain and a region in FGFR1 kinase domain in a phosphorylation independent manner. These experiments reveal a mechanism for how SH2 domain selectivity is regulated in vivo to mediate a specific cellular process.