Takeo Tsuda

Lumenal gating mechanism revealed in calcium pump crystal structures with phosphate analogues

P-type ion transporting ATPases are ATP-powered ion pumps that establish ion concentration gradients across biological membranes. Transfer of bound cations to the lumenal or extracellular side occurs while the ATPase is phosphorylated. Here we report at 2.3 Å resolution the structure of the calcium-ATPase of skeletal muscle sarcoplasmic reticulum, a representative P-type ATPase that is crystallized in the absence of Ca2+ but in the presence of magnesium fluoride, a stable phosphate analogue. This and other crystal structures determined previously provide atomic models for all four principal states in the reaction cycle. These structures show that the three cytoplasmic domains rearrange to move six out of ten transmembrane helices, thereby changing the affinity of the Ca2+-binding sites and the gating of the ion pathway. Release of ADP triggers the opening of the lumenal gate and release of phosphate its closure, effected mainly through movement of the A-domain, the actuator of transmembrane gates.

Modeling of the inhibitory interaction of phospholamban with the Ca2+ ATPase

The inhibitory interaction of phospholamban (PLN) with the sarco(endo)plasmic reticulum Ca2+ ATPase isoform 1 (SERCA1a) was modeled on the basis of several constraints which included (i) spontaneous formation of SS-bridges between mutants L321C in transmembrane helix 4 (M4) of SERCA1a and N27C in PLN and between V89C (M4) and V49C (PLN); (ii) definition of the face of the PLN transmembrane helix that interacts with SERCA; (iii) cross-linking between Lys-3 of PLN and Lys-397 and Lys-400 of SERCA2a. The crystal structure of SERCA1a in the absence of Ca2+, which binds PLN, was used as the structure into which an atomic model of PLN was built. PLN can fit into a transmembrane groove formed by the juxtaposition of M2, the upper part of M4, M6, and M9. In the SERCA1a structure with bound Ca2+, this groove is closed, accounting for the ability of Ca2+ to disrupt PLN–SERCA interactions. Near the cytoplasmic surface of the bilayer, the PLN helix is disrupted to prevent its collision with M4. The model can be extended into the cytoplasmic domain so that Lys-3 in PLN can be cross-linked with Lys-397 and Lys-400 in SERCA1a with little unwinding of the N-terminal helix of PLN.

How processing of aspartylphosphate is coupled to lumenal gating of the ion pathway in the calcium pump

Ca2+-ATPase of skeletal muscle sarcoplasmic reticulum is the best-studied member of the P-type or E1/E2 type ion transporting ATPases. It has been crystallized in seven different states that cover nearly the entire reaction cycle. Here we describe the structure of this ATPase complexed with phosphate analogs BeF3− and AlF4− in the absence of Ca2+, which correspond to the E2P ground state and E2∼P transition state, respectively. The luminal gate is open with BeF3− and closed with AlF4−. These and the E1∼P·ADP analog crystal structures show that a two-step rotation of the cytoplasmic A-domain opens and closes the luminal gate through the movements of the M1–M4 transmembrane helices. There are several conformational switches coupled to the rotation, and the one in the cytoplasmic part of M2 has critical importance. In the second step of rotation, positioning of one water molecule couples the hydrolysis of aspartylphosphate to closing of the gate.

The Asn-420-linked sugar chain in human epidermal growth factor receptor suppresses ligand-independent spontaneous oligomerization. Possible role of a specific sugar chain in controllable receptor activation.

To elucidate a role(s) of Asn-linked sugar chain(s) in the function of epidermal growth factor receptor (EGFR), a series of the EGFR mutants were prepared in which potential glycosylation sites in the domain III were eliminated by site-directed mutagenesis. Although the wild-type and mutants of Asn-328, Asn-337, and Asn-389 underwent autophosphorylation in response to epidermal growth factor (EGF), the Asn-420 → Gln mutant was found to be constitutively tyrosine-phosphorylated. This abnormal ligand-independent phosphorylation of the mutant appears to be due to a ligand-independent spontaneous oligomer formation, as shown by a cross-linking experiment using the purified soluble extracellular domain (sEGFR). As revealed by the dissociation of the Asn-420 → Gln sEGFR oligomer by simple dilution, it seems likely that the equilibrium is shifted toward oligomer formation to an unusual degree. Furthermore, it was also found that the mutation caused a loss of the ability to bind EGF. These findings suggest that the sugar chain linked to Asn-420 plays a crucial role in EGF binding and prevents spontaneous oligomerization of the EGFR, which may otherwise lead to uncontrollable receptor activation, and support the view of a specific role of an Asn-linked sugar chain in the function of a glycoprotein.

Role of N-glycans in growth factor signaling

Secreted proteins and membrane proteins are frequently post-translationally modified by oligosaccharides. Therefore, many glycoproteins are involved in signal transduction. One example is growth factor receptors, which are membrane proteins that often contain oligosaccharides. The oligosaccharides in those growth factor receptors play crucial roles in receptor functions. An analysis of glycosyltransferase-transfectants revealed that the branching structures of oligosaccharide also serve as important determinants. For example, N-glycans of epidermal growth factor receptor (EGFR) are involved in receptor sorting, ligand binding and dimerization. The addition of a bisecting GlcNAc to N-glycans increases the endocytosis of EGFR. N-glycans of Trk, a high affinity nerve growth factor receptor, also affect its function. Thus, oligosaccharides play an important role in growth factor signaling. Published in 2004.

The Critical Role of the Stem Region as a Functional Domain Responsible for the Oligomerization and Golgi Localization of N-Acetylglucosaminyltransferase V THE INVOLVEMENT OF A DOMAIN HOMOPHILIC INTERACTION

We demonstrated that a region in the stem ofN-acetylglucosaminyltransferase V (GnT-V), a Golgi resident protein, is not required for enzyme activity but serves as functional domain, responsible for intracellular localization. Deletion of the domain led to complete retention of the kinetic properties but resulted in the cell surface localization of the enzyme as well as its efficient secretion into the medium. The lack of this domain concomitantly abolished the disulfide-mediated oligomerization of GnT-V, which appears to confer the Golgi retention. When the domain was inserted into the stem region of a cell surface-localized type II membrane protein, the resulting chimeric protein was substantially oligomerized and predominantly localized in the intracellular organelle. Furthermore, it was found that the presence of this domain is exclusively responsible for homo-oligomer formation. This homophilic interaction appears to involve a hydrophobic cluster of residues in the α-helix of the domain, as indicated by secondary structure predictions. These findings suggest that the domain specifically participates in the Golgi retention of GnT-V, probably via inducing homo-oligomer formation, and would also provide a possible mechanism for the oligomerization, which is critical for localization in the Golgi.

Domain organization and movements in heavy metal ion pumps : Papain digestion of copa, A Cu+ -TRANSPORTING ATPase

To study domain organization and movements in the reaction cycle of heavy metal ion pumps, CopA, a bacterial Cu+-ATPase from Thermotoga maritima was cloned, overexpressed, and purified, and then subjected to limited proteolysis using papain. Stable analogs of intermediate states were generated using AMPPCP as a nonhydrolyzable ATP analog and AlFx as a phosphate analog, following conditions established for Ca2+-ATPase (SERCA1). Characteristic digestion patterns obtained for different analog intermediates show that CopA undergoes domain rearrangements very similar to those of SERCA1. Digestion sites were identified on the loops connecting the A-domain and the transmembrane helices M2 and M3 as well as on that connecting the N-terminal metal binding domain (NMBD) and the first transmembrane helix, Ma. These digestion sites were protected in the E1P·ADP and E2P analogs, whereas the M2–A-domain loop was cleaved specifically in the absence of ions to be transported, just as in SERCA1. ATPase activity was lost when the link between the NMBD and the transmembrane domain was cleaved, indicating that the NMBD plays a critical role in ATP hydrolysis in T. maritima CopA. The change in susceptibility of the loop between the NMBD and Ma helix provides evidence that the NMBD is associated to the A-domain and recruited into domain rearrangements and that the Ma helix is the counterpart of the M1 helix in SERCA1 and Mb and Mc are uniquely inserted before M2.

Nucleotide recognition by CopA, a Cu+-transporting P-type ATPase.

Heavy metal pumps constitute a large subgroup in P‐type ion‐transporting ATPases. One of the outstanding features is that the nucleotide binding N‐domain lacks residues critical for ATP binding in other well‐studied P‐type ATPases. Instead, they possess an HP‐motif and a Gly‐rich sequence in the N‐domain, and their mutations impair ATP binding. Here, we describe 1.85 Å resolution crystal structures of the P‐ and N‐domains of CopA, an archaeal Cu+‐transporting ATPase, with bound nucleotides. These crystal structures show that CopA recognises the adenine ring completely differently from other P‐type ATPases. The crystal structure of the His462Gln mutant, in the HP‐motif, a disease‐causing mutation in human Cu+‐ATPases, shows that the Gln side chain mimics the imidazole ring, but only partially, explaining the reduction in ATPase activity. These crystal structures lead us to propose a role of the His and a mechanism for removing Mg2+ from ATP before phosphoryl transfer.

ATP and Acetyl Phosphate Induces Molecular Events near the ATP Binding Site and the Membrane Domain of Na+,K+-ATPase THE TETRAMERIC NATURE OF THE ENZYME

The addition of ATP to Mg2+-Na+-bound-probe labeled Na+,K+-ATPase preparations containing ∼0.5 mol of pyridoxal 5′-diphospho-5′-adenosine (AP2PL) probe at Lys-480 and ∼0.9 mol of fluorescein 5′-isothiocyanate (FITC) probe at Lys-501 showed a decrease and an increase in the AP2PL fluorescence intensity with neither significant ATP-dependent phosphorylation nor FITC fluorescence change. The rate constants for the fluorescence change increased nearly linearly with increasing ATP concentrations. The substitution of AcP for ATP decreased the FITC fluorescence rather monophasically, 8.5/s, which was followed by the half-site phosphorylation with same amount of components with different rate constant, 7.2 and 4.6/s, followed by a much slower increase in the two components of AP2PL fluorescence, 1.4 and 0.2/s. The addition of Na+ with increasing concentrations of ATP to the K+-bound AP2PL-FITC enzymes induced accelerations in the decrease and an increase in the AP2PL fluorescence intensity with two different increases in the FITC fluorescence intensity, showing that the same concentration of ATP is capable of inducing four different fluorescence changes. The addition of ATP to the Mg2+-Na+-bound enzymes modified withN-[p-(2-benzimidazolyl)phenyl]-maleimide (BIPM) at Cys-964 and retaining full Na+,K+-ATPase activity induced two different increases in BIPM fluorescence intensity. Each rate constant for the BIPM fluorescence change versus concentrations of ATP gave two intersecting straight lines. These data and the stoichiometries of fluorescence probe bindings and ATP- and AcP-dependent phosphorylation provide strong support for the conclusion that the functional membrane-bound Na+,K+-ATPase is a tetramer.

The action of N-acetylglucosaminyltransferase-V is prevented by the bisecting GlcNAc residue at the catalytic step

Using a purified protein and bisected acceptor oligosaccharides, we demonstrate that N‐acetylglucosaminyltransferase (GnT)‐V transfers a N‐acetylglucosamine residue via a β1,6‐linkage to the bisected oligosaccharides. We also kinetically characterized the substrate specificity of GnT‐V with respect to the bisected oligosaccharide. Although the K m values for the bisected acceptors were comparable to that for a non‐bisected acceptor, the V max values for the bisected acceptors were much lower than that for the non‐bisected acceptor. These findings suggest that the acceptor specificity of GnT‐V is determined by the catalytic process rather than by its binding to the substrate. It was also found that the presence of the 2‐N‐acetyl group in the bisecting monosaccharide moiety plays a critical role in determining the catalytic efficiency of the enzyme.