Douglas M. Freymann

Conserved tryptophan in the core domain of transglutaminase is essential for catalytic activity

Transglutaminase 2 (TG2) is a distinctive member of the family of Ca2+-dependent enzymes recognized mostly by their abilities to catalyze the posttranslational crosslinking of proteins. TG2 uniquely binds and hydrolyzes GTP; binding GTP inhibits its crosslinking activity but allows it to function in signal transduction (hence the Gh designation). The core domain of TG2 (residues 139–471, rat) comprises the papain-like catalytic triad and the GTP-binding domain (residues 159–173) and contains almost all of the conserved tryptophans of the protein. Examining point mutations at Trp positions 180, 241, 278, 332, and 337 showed that, upon binding 2′-(or 3′)-O-(N-methylanthraniloyl)GTP (mantGTP), the Phe-332 mutant was the weakest (35% less than wild type) in resonance energy transfer from the protein (λexc, max = 290 nm) to the mant fluorophore (λem = 444 nm) and had a reduced affinity for mantGTP. Trp-332, situated near the catalytic center and the nucleotide-binding area of TG2, may be part of the allosteric relay machinery that transmits negative effector signals from nucleotide binding to the active center of TG2. A most important observation was that, whereas no enzyme activity could be detected when Trp-241 was replaced with Ala or Gln, partial preservation of catalytic activity was seen with substitutions by Tyr > Phe > His. The results indicate that Trp-241 is essential for catalysis, possibly by stabilizing the transition states by H-bonding, quadrupole–ion, or van der Waals interactions. This contrasts with the evolutionarily related papain family of cysteine proteases, which uses Gln-19 (papain) for stabilizing the transition state.

Structural basis for mobility in the 1.1 A crystal structure of the NG domain of Thermus aquaticus Ffh.

The NG domain of the prokaryotic signal recognition protein Ffh is a two-domain GTPase that comprises part of the prokaryotic signal recognition particle (SRP) that functions in co-translational targeting of proteins to the membrane. The interface between the N and G domains includes two highly conserved sequence motifs and is adjacent in sequence and structure to one of the conserved GTPase signature motifs. Previous structural studies have shown that the relative orientation of the two domains is dynamic. The N domain of Ffh has been proposed to function in regulating the nucleotide-binding interactions of the G domain. However, biochemical studies suggest a more complex role for the domain in integrating communication between signal sequence recognition and interaction with receptor. Here, we report the structure of the apo NG GTPase of Ffh from Thermus aquaticus refined at 1.10 A resolution. Although the G domain is very well ordered in this structure, the N domain is less well ordered, reflecting the dynamic relationship between the two domains previously inferred. We demonstrate that the anisotropic displacement parameters directly visualize the underlying mobility between the two domains, and present a detailed structural analysis of the packing of the residues, including the critical alpha4 helix, that comprise the interface. Our data allows us to propose a structural explanation for the functional significance of sequence elements conserved at the N/G interface.

Structural coupling of the EF hand and C‐terminal GTPase domains in the mitochondrial protein Miro

Miro is a highly conserved calcium‐binding GTPase at the regulatory nexus of mitochondrial transport and autophagy. Here we present crystal structures comprising the tandem EF hand and carboxy terminal GTPase (cGTPase) domains of Drosophila Miro. The structures reveal two previously unidentified ‘hidden’ EF hands, each paired with a canonical EF hand. Each EF hand pair is bound to a helix that structurally mimics an EF hand ligand. A key nucleotide‐sensing element and a Pink1 phosphorylation site both lie within an extensive EF hand–cGTPase interface. Our results indicate structural mechanisms for calcium, nucleotide and phosphorylation‐dependent regulation of mitochondrial function by Miro.

X‐ray structure of the T. Aquaticus Ftsy:GDP complex suggests functional roles for the C‐terminal helix of the SRP GTPases

FtsY and Ffh are structurally similar prokaryotic Signal Recognition Particle GTPases that play an essential role in the Signal Recognition Particle (SRP)‐mediated cotranslational targeting of proteins to the membrane. The two GTPases assemble in a GTP‐dependent manner to form a heterodimeric SRP targeting complex. We report here the 2.1 Å X‐ray structure of FtsY from T. aquaticus bound to GDP. The structure of the monomeric protein reveals, unexpectedly, canonical binding interactions for GDP. A comparison of the structures of the monomeric and complexed FtsY NG GTPase domain suggests that it undergoes a conformational change similar to that of Ffh NG during the assembly of the symmetric heterodimeric complex. However, in contrast to Ffh, in which the C‐terminal helix shifts independently of the other subdomains, the C‐terminal helix and N domain of T. aquaticus FtsY together behave as a rigid body during assembly, suggesting distinct mechanisms by which the interactions of the NG domain “module” are regulated in the context of the two SRP GTPases. Proteins 2007.

Novel protein and Mg2+ configurations in the Mg2+GDP complex of the SRP GTPase ffh

Ffh is the signal sequence recognition and targeting subunit of the prokaryotic signal recognition particle (SRP). Previous structural studies of the NG GTPase domain of Ffh demonstrated magnesium‐dependent and magnesium‐independent binding conformations for GDP and GMPPNP that are believed to reflect novel mechanisms for exchange and activation in this member of the GTPase superfamily. The current study of the NG GTPase bound to Mg2+GDP reveals two new binding conformations—in the first the magnesium interactions are similar to those seen previously, however, the protein undergoes a conformational change that brings a conserved aspartate into its second coordination sphere. In the second, the protein conformation is similar to that seen previously, but the magnesium coordination sphere is disrupted so that only five oxygen ligands are present. The loss of the coordinating water molecule, at the position that would be occupied by the oxygen of the γ‐phosphate of GTP, is consistent with that position being privileged for exchange during phosphate release. The available structures of the GDP‐bound protein provide a series of structural snapshots that illuminate steps along the pathway of GDP release following GTP hydrolysis. Proteins 2004;54:000–000.

Structural insights into Parkin substrate lysine targeting from minimal Miro substrates

Hereditary Parkinson’s disease is commonly caused by mutations in the protein kinase PINK1 or the E3 ubiquitin ligase Parkin, which function together to eliminate damaged mitochondria. PINK1 phosphorylates both Parkin and ubiquitin to stimulate ubiquitination of dozens of proteins on the surface of the outer mitochondrial membrane. However, the mechanisms by which Parkin recognizes specific proteins for modification remain largely unexplored. Here, we show that the C-terminal GTPase (cGTPase) of the Parkin primary substrate human Miro is necessary and sufficient for efficient ubiquitination. We present several new X-ray crystal structures of both human Miro1 and Miro2 that reveal substrate recognition and ubiquitin transfer to be specific to particular protein domains and lysine residues. We also provide evidence that Parkin substrate recognition is functionally separate from substrate modification. Finally, we show that prioritization for modification of a specific lysine sidechain of the cGTPase (K572) within human Miro1 is dependent on both its location and chemical microenvironment. Activation of Parkin by phosphorylation or by binding of pUb is required for prioritization of K572 for modification, suggesting that Parkin activation and acquisition of substrate specificity are coupled.

Crystallization of the GMPPCP complex of the NG domains of Thermus aquaticus Ffh and FtsY.

The GTPases Ffh and FtsY are components of the prokaryotic signal recognition particle protein-targeting pathway. The two proteins interact in a GTP-dependent manner, forming a complex that can be stabilized by use of the non-hydrolyzable GTP analog GMPPCP. Crystals of the complex of the NG GTPase domains of the two proteins have been obtained from ammonium sulfate solutions. Crystals grow with several different morphologies, predominately as poorly diffracting plates and needle clusters, but occasionally as well diffracting rods. It has been demonstrated that all forms of the crystals observed contain an intact complex. Diffraction data to 2.0 A resolution have been measured.

Mapping the functional anatomy of Orai1 transmembrane domains for CRAC channel gating

Significance Store-operated Orai1 channels mediate transcriptional, proliferative, and effector-cell programs in many cells. Mutations in Orai1 that block channel activation or evoke constitutive channel activity are known to cause debilitating diseases in humans such as immunodeficiency, autoimmunity, myopathy, and thrombocytopenia. However, our understanding of the underlying molecular mechanisms of these diseases is limited by fundamental gaps in how Orai1 channels are gated. Here, we map key functional interactions between the transmembrane domains of Orai1 and identify several contacts that are critical for conveying the STIM1 gating signal to the pore. Our findings illuminate important allosteric interactions between topologically distinct domains of Orai1 and help elucidate the molecular underpinnings of disease-causing mutations. Store-operated Orai1 channels are activated through a unique inside-out mechanism involving binding of the endoplasmic reticulum Ca2+ sensor STIM1 to cytoplasmic sites on Orai1. Although atomic-level details of Orai structure, including the pore and putative ligand binding domains, are resolved, how the gating signal is communicated to the pore and opens the gate is unknown. To address this issue, we used scanning mutagenesis to identify 15 residues in transmembrane domains (TMs) 1–4 whose perturbation activates Orai1 channels independently of STIM1. Cysteine accessibility analysis and molecular-dynamics simulations indicated that constitutive activation of the most robust variant, H134S, arises from a pore conformational change that opens a hydrophobic gate to augment pore hydration, similar to gating evoked by STIM1. Mutational analysis of this locus suggests that H134 acts as steric brake to stabilize the closed state of the channel. In addition, atomic packing analysis revealed distinct functional contacts between the TM1 pore helix and the surrounding TM2/3 helices, including one set mediated by a cluster of interdigitating hydrophobic residues and another by alternative ridges of polar and hydrophobic residues. Perturbing these contacts via mutagenesis destabilizes STIM1-mediated Orai1 channel gating, indicating that these bridges between TM1 and the surrounding TM2/3 ring are critical for conveying the gating signal to the pore. These findings help develop a framework for understanding the global conformational changes and allosteric interactions between topologically distinct domains that are essential for activation of Orai1 channels.

Nucleotide-binding flexibility in ultrahigh-resolution structures of the SRP GTPase Ffh

Crystal structures of the Ffh NG GTPase domain at < 1.24 Å resolution reveal multiple overlapping nucleotide binding modes.

Structural Coupling of the EF Hand and C-Terminal GTPase Domains in the Mitochondrial Protein Miro

The outer mitochondrial membrane protein Miro is a highly conserved calcium-binding GTPase that is at the regulatory nexus of several processes, including mitochondrial transport and autophagy. Miro attaches mitochondria to the microtubule-based motor protein kinesin-1 and acts as a calcium-dependent switch for mitochondrial movement. Phosphorylation of Miro by Pink1 kinase and its subsequent Parkin-mediated degradation leads to mitophagy of damaged mitochondria. Relatively little is known about the molecular underpinnings of these processes and a structural understanding of the relevant protein machinery is lacking. Here we present crystal structures comprising the tandem EF hand and C-terminal GTPase (cGTPase) domains of Drosophila Miro. The structures reveal two previously unidentified “hidden” EF hands, each paired with a canonical EF hand. Each EF hand pair is bound to a helix that structurally mimics an EF hand ligand. A key nucleotide-sensing element and a Pink1 phosphorylation site both lie within an extensive EF hand/cGTPase interface and may have implications for Pink1-mediated recruitment of Parkin to the mitochondrial surface. Our results suggest structural mechanisms for calcium, nucleotide, and phosphorylation-dependent regulation of mitochondrial function by Miro.