Oedema factor, a calmodulin-activated adenylyl cyclase, is important in the pathogenesis of anthrax. Here we report the X-ray structures of oedema factor with and without bound calmodulin. Oedema ...factor shares no significant structural homology with mammalian adenylyl cyclases or other proteins. In the active site, 3'-deoxy-ATP and a single metal ion are well positioned for catalysis with histidine 351 as the catalytic base. This mechanism differs from the mechanism of two-metal-ion catalysis proposed for mammalian adenylyl cyclases. Four discrete regions of oedema factor form a surface that recognizes an extended conformation of calmodulin, which is very different from the collapsed conformation observed in other structures of calmodulin bound to effector peptides. On calmodulin binding, an oedema factor helical domain of relative molecular mass 15,000 undergoes a 15 A translation and a 30 degrees rotation away from the oedema factor catalytic core, which stabilizes a disordered loop and leads to enzyme activation. These allosteric changes provide the first molecular details of how calmodulin modulates one of its targets.
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Dostopno za:
DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The calcium binding proteins of the EF-hand super-family are involved in the regulation of all aspects of cell function. These proteins exhibit a great diversity of composition, structure, Ca
...2+-binding and target interaction properties. Here, our current understanding of the Ca
2+-binding mechanism is assessed. The structures of the EF-hand motifs containing 11–14 amino acid residues in the Ca
2+-binding loop are analyzed within the framework of the recently proposed two-step Ca
2+-binding mechanism. A hypothesis is put forward that in all EF-hand proteins the Ca
2+-binding and the resultant conformational responses are governed by the central structure connecting the Ca
2+-binding loops in the two-EF-hand domain. This structure, named EFβ-scaffold, defines the position of the bound Ca
2+, and coordinates the function of the N-terminal (variable and flexible) with the C-terminal (invariable and rigid) parts of the Ca
2+-binding loop. It is proposed that the nature of the first ligand of the Ca
2+-binding loop is an important determinant of the conformational change. Additional factors, including the interhelical contacts, the length, structure and flexibility of the linker connecting the EF-hand motifs, and the overall energy balance provide the fine-tuning of the Ca
2+-induced conformational change in the EF-hand proteins.
Calmodulin (CaM), a member of the EF-hand superfamily, regulates many aspects of cell function by responding specifically to micromolar concentrations of Ca2+ in the presence of an ∼1000-fold higher ...concentration of cellular Mg2+. To explain the structural basis of metal ion binding specificity, we have determined the X-ray structures of the N-terminal domain of calmodulin (N-CaM) in complexes with Mg2+, Mn2+, and Zn2+. In contrast to Ca2+, which induces domain opening in CaM, octahedrally coordinated Mg2+ and Mn2+ stabilize the closed-domain, apo-like conformation, while tetrahedrally coordinated Zn2+ ions bind at the protein surface and do not compete with Ca2+. The relative positions of bound Mg2+ and Mn2+ within the EF-hand loops are similar to those of Ca2+; however, the Glu side chain at position 12 of the loop, whose bidentate interaction with Ca2+ is critical for domain opening, does not bind directly to either Mn2+ or Mg2+, and the vacant ligand position is occupied by a water molecule. We conclude that this critical interaction is prevented by specific stereochemical constraints imposed on the ligands by the EF-hand β-scaffold. The structures suggest that Mg2+ contributes to the switching off of calmodulin activity and possibly other EF-hand proteins at the resting levels of Ca2+. The Mg2+-bound N-CaM structure also provides a unique view of a transiently bound hydrated metal ion and suggests a role for the hydration water in the metal-induced conformational change.
Calmodulin (CaM) is a multifunctional Ca
2+-binding protein that regulates the activity of many enzymes in response to changes in the intracellular Ca
2+ concentration. There are two globular domains ...in CaM, each containing a pair of helix-loop-helix Ca
2+-binding motifs called EF-hands. Ca
2+-binding induces the opening of both domains thereby exposing hydrophobic pockets that provide binding sites for the target enzymes. Here, I present a 2.4
Å resolution structure of a calmodulin mutant (CaM41/75) in which the N-terminal domain is locked in the closed conformation by a disulfide bond. CaM41/75 crystallized in a tetragonal lattice with the Ca
2+ bound in all four EF-hands. In the closed N-terminal domain Ca ions are coordinated by the four protein ligands in positions 1, 3, 5 and 7 of the loop, and by two solvent ligands. The glutamate side-chain in the 12th position of the loop (Glu31 in site I and Glu67 in site II), which in the wild-type protein provides a bidentate Ca
2+ ligand, remains in a distal position. Based on a comparison of CaM41/75 with other CaM and troponin C structures a detailed two-step mechanism of the Ca
2+-binding process is proposed. Initially, the Ca
2+ binds to the N-terminal part of the loop, thus generating a rigid link between the incoming helix (helix A, or helix C) and the central beta structure involving the residues in the sixth, seventh and eighth position of the loop. Then, the exiting helix (helix B or helix D) rotates causing the glutamate ligand in the 12th position to move into the vicinity of the immobilized Ca
2+. An adjustment of the ϕ, ψ backbone dihedral angles of the Ile residue in the eighth position is necessary and sufficient for the helix rotation and functions as a hinge. The model allows for a significant independence of the Ca
2+-binding sites in a two-EF-hand domain.
The activation mechanism of Ca(2+)/calmodulin-dependent protein kinase II (alphaCaMKII) is investigated by steady-state and stopped-flow fluorescence spectroscopies. Lys(75)-labeled TA-cal Török, K., ...and Trentham, D. R. (1994) Biochemistry 33, 12807-12820 is used to measure binding events, and double-labeled AEDANS,DDP-T34C/T110/C-calmodulin Drum et al. (2000) J. Biol. Chem. 275, 36334-36340 (DA-cal) is used to detect changes in calmodulin conformation. Fluorescence quenching of DA-cal attributed to resonance energy transfer is related to the compactness of the calmodulin molecule. Interprobe distances are estimated by lifetime measurements of Ca(2+)/DA-cal in complexes with unphosphorylated nucleotide-free, nucleotide-bound, and Thr(286)-phospho-alphaCaMKII as well as with alphaCaMKII-derived calmodulin-binding peptides in the presence of Ca(2+). These measurements show that calmodulin can assume at least two spectrally distinct conformations when bound to alphaCaMKII with estimated interprobe distances of 40 and 22-26 A. Incubation with ATP facilitates the assumption of the most compact conformation. Nonhydrolyzable ATP analogues partially replicate the effects of ATP, suggesting that while the binding of ATP induces a conformational change, Thr(286)-autophosphorylation is probably required for the transition of calmodulin into its most compact conformer. The rate constant for the association of Ca(2+)/TA-cal with alphaCaMKII is estimated as 2 x 10(7) M(-1) s(-1) and is not substantially affected by the presence of ATP. The rate of net calmodulin compaction measured by Ca(2+)/DA-cal is markedly slower, occurring with a rate constant of 2.5 x 10(6) M(-1) s(-1), suggesting that unproductive complexes may play a role in the activation mechanism.
Wiskott-Aldrich syndrome protein (WASP)-homology domain 2 (WH2) is a small and widespread actin-binding motif. In the WASP family, WH2 plays a role in filament nucleation by Arp2/3 complex. Here we ...describe the crystal structures of complexes of actin with the WH2 domains of WASP, WASP-family verprolin homologous protein, and WASP-interacting protein. Despite low sequence identity, WH2 shares structural similarity with the N-terminal portion of the actin monomer-sequestering thymosin β domain (Tβ). We show that both domains inhibit nucleotide exchange by targeting the cleft between actin subdomains 1 and 3, a common binding site for many unrelated actin-binding proteins. Importantly, WH2 is significantly shorter than Tβ but binds actin with ≈10-fold higher affinity. WH2 lacks a C-terminal extension that in Tβ4 becomes involved in monomer sequestration by interfering with intersubunit contacts in F-actin. Owing to their shorter length, WH2 domains connected in tandem by short linkers can coexist with intersubunit contacts in F-actin and are proposed to function in filament nucleation by lining up actin subunits along a filament strand. The WH2-central region of WASP-family proteins is proposed to function in an analogous way by forming a special class of tandem repeats whose function is to line up actin and Arp2 during Arp2/3 nucleation. The structures also suggest a mechanism for how profilin-binding Pro-rich sequences positioned N-terminal to WH2 could feed actin monomers directly to WH2, thereby playing a role in filament elongation.
The edema factor exotoxin produced byBacillus anthracis is an adenylyl cyclase that is activated by calmodulin (CaM) at resting state calcium concentrations in infected cells. A C-terminal 60-kDa ...fragment corresponding to the catalytic domain of edema factor (EF3) was cloned, overexpressed inEscherichia coli, and purified. The N-terminal 43-kDa domain (EF3-N) of EF3, the sole domain of edema factor homologous to adenylyl cyclases from Bordetella pertussis andPseudomonas aeruginosa, is highly resistant to protease digestion. The C-terminal 160-amino acid domain (EF3-C) of EF3 is sensitive to proteolysis in the absence of CaM. The addition of CaM protects EF3-C from being digested by proteases. EF3-N and EF3-C were expressed separately, and both fragments were required to reconstitute full CaM-sensitive enzyme activity. Fluorescence resonance energy transfer experiments using a double-labeled CaM molecule were performed and indicated that CaM adopts an extended conformation upon binding to EF3. This contrasts sharply with the compact conformation adopted by CaM upon binding myosin light chain kinase and CaM-dependent protein kinase type II. Mutations in each of the four calcium binding sites of CaM were examined for their effect on EF3 activation. Sites 3 and 4 were found critical for the activation, and neither the N- nor the C-terminal domain of CaM alone was capable of activating EF3. A genetic screen probing loss-of-function mutations of EF3 and site-directed mutations based on the homology of the edema factor family revealed a conserved pair of aspartate residues and an arginine that are important for catalysis. Similar residues are essential for di-metal-mediated catalysis in mammalian adenylyl cyclases and a family of DNA polymerases and nucleotidyltransferases. This suggests that edema factor may utilize a similar catalytic mechanism.
Structure of the Light Chain-Binding Domain of Myosin V Terrak, Mohammed; Rebowski, Grzegorz; Lu, Renne C. ...
Proceedings of the National Academy of Sciences - PNAS,
09/2005, Letnik:
102, Številka:
36
Journal Article, Web Resource
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Myosin V is a double-headed molecular motor involved in organelle transport. Two distinctive features of this motor, processivity and the ability to take extended linear steps of ≈36 nm along the ...actin helical track, depend on its unusually long light chain-binding domain (LCBD). The LCBD of myosin V consists of six tandem IQ motifs, which constitute the binding sites for calmodulin (CaM) and Cam-like light chains. Here, we report the 2-Å resolution crystal structure of myosin light chain 1 (Mlc1p) bound to the IQ2-IQ3 fragment of Myo2p, a myosin V from Saccharomyces cerevisiae. This structure, combined with FRET distance measurements between probes in various CaM-IQ complexes, comparative sequence analysis, and the previously determined structures of Mlc1p-IQ2 and Mlc1p-IQ4, allowed building a model of the LCBD of myosin V. The IQs of myosin V are distributed into three pairs. There appear to be specific cooperative interactions between light chains within each IQ pair, but little or no interaction between pairs, providing flexibility at their junctions. The second and third IQ pairs each present a light chain, whether CaM or a CaM-related molecule, bound in a noncanonical extended conformation in which the N-lobe does not interact with the IQ motif. The resulting free N-lobes may engage in protein-protein interactions. The extended conformation is characteristic of the single IQ of myosin VI and is common throughout the myosin superfamily. The model points to a prominent role of the LCBD in the function, regulation, and molecular interactions of myosin V.
A two-step zero-length crosslinking procedure for studying protein-protein complexes has been developed. One component of a complex is briefly incubated with ...1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) in the presence of N-hydroxysuccinimide resulting in the conversion of some of the protein carboxyls into succinimidyl esters. The reaction is stopped by addition of beta-mercaptoethanol and other interacting proteins are then added. Crosslinking arises from substitution of lysine epsilon-amino groups of these proteins for the succinimidyl moieties during a 1- to 2-h incubation period. The advantage of this method versus one-step zero-length crosslinking is that only one component of the complex is exposed to the crosslinker, which eliminates complications arising from the formation of crosslinks among several proteins of a multicomponent complex. Furthermore, crosslinks can be formed even in the presence of reagents, such as dithiothreitol and EDTA, that would interfere with direct crosslinking with EDC.
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