The solution structure of apocalmodulin from Saccharomyces cerevisiae implies a mechanism for its unique Ca2+ binding property

We have determined the solution structure of calmodulin (CaM) from yeast (Saccharomyces cerevisiae) (yCaM) in the apo state by using NMR spectroscopy. yCaM is 60% identical in its amino acid sequence with other CaMs, and exhibits its unique biological features. yCaM consists of two similar globular domains (N- and C-domain) containing three Ca(2+)-binding motifs, EF-hands, in accordance with the observed 3 mol of Ca(2+) binding. In the solution structure of yCaM, the conformation of the N-domain conforms well to the one of the expressed N-terminal half-domains of yCaM [Ishida, H., et al. (2000) Biochemistry 39, 13660-13668]. The conformation of the C-domain basically consists of a pair of helix-loop-helix motifs, though a segment corresponding to the forth Ca(2+)-binding site of CaM deviates in its primary structure from a typical EF-hand motif and loses the ability to bind Ca(2+). Thus, the resulting conformation of each domain is essentially identical to the corresponding domain of CaM in the apo state. A flexible linker connects the two domains as observed for CaM. Any evidence for the previously reported interdomain interaction in yCaM was not observed in the solution structure of the apo state. Hence, the interdomain interaction possibly occurs in the course of Ca(2+) binding and generates a cooperative Ca(2+) binding among all three sites. Preliminary studies on a mutant protein of yCaM, E104Q, revealed that the Ca(2+)-bound N-domain interacts with the apo C-domain and induces a large conformational change in the C-domain.     

Role of the N-terminal region of the skeletal muscle myosin light chain kinase target sequence in its interaction with calmodulin.

The binding of calmodulin (CaM) to four synthetic peptide analogues of the skeletal muscle myosin light chain kinase (sk-MLCK) target sequence has been studied using 1H-NMR. The 18-residue peptide WFF is anchored to CaM via the interaction of the Trp 4 side chain with the C-domain and the Phe 17 side chain with the N-domain of the protein. A peptide corresponding to the first 10 residues (WF10) does not provide the second anchoring residue and is not long enough to span both domains of CaM. 1H-NMR spectroscopy indicates that the WF10 peptide interacts specifically with the C-domain of CaM, and the chemical shifts of the bound Trp side chain are very similar in the CaM:WF10 and CaM:WFF complexes. Binding of the C-domain of CaM to the strongly basic region around Trp 4 of this MLCK sequence may be an important step in target recognition. Comparison of 1H-NMR spectra of CaM bound to WFF, a Trp 4-->Phe analogue (FFF), or a Trp 4-->Phe/Phe 17-->Trp analogue (FFW) suggests that all three peptides bind to CaM in the same orientation, i.e., with the peptide side chain in position 4 interacting with the C-domain and the side chain in position 17 interacting with the N-domain. This indicates that a Trp residue in position 4 is not an absolute requirement for binding this target sequence and that interchanging the Trp 4 and Phe 17 residues does not reverse the orientation of the bound peptide, in confirmation of the deduction from previous indirect studies using circular dichroism (Findlay WA, Martin SR, Beckingham K, Bayley PM, 1995, Biochemistry 34:2087-2094). Molecular modeling/energy minimization studies indicate that only minor local changes in the protein structure are required to accommodate binding of the bulkier Trp 17 side chain of the FFW peptide to the N-domain of CaM.     

The NMDA receptor NR1 C1 region bound to calmodulin: structural insights into functional differences between homologous domains

Calmodulin (CaM) regulates tetrameric N-methyl-D-aspartate receptors (NMDARs) by binding tightly to the C0 and C1 regions of its NR1 subunit. A crystal structure (2HQW; 1.96 A) of calcium-saturated CaM bound to NR1C1 (peptide spanning 875-898) showed that NR1 S890, whose phosphorylation regulates membrane localization, was solvent protected, whereas the endoplasmic reticulum retention motif was solvent exposed. NR1 F880 filled the CaM C-domain pocket, whereas T886 was closest to the N-domain pocket. This 1-7 pattern was most similar to that in the CaM-MARCKS complex. Comparison of CaM-ligand wrap-around conformations identified a core tetrad of CaM C-domain residues (FLMM(C)) that contacted all ligands consistently. An identical tetrad of N-domain residues (FLMM(N)) made variable sets of contacts with ligands. This CaM-NR1C1 structure provides a foundation for designing mutants to test the role of CaM in NR1 trafficking as well as insights into how the homologous CaM domains have different roles in molecular recognition.     

Structure of the complex of calmodulin with the target sequence of calmodulin-dependent protein kinase I: studies of the kinase activation mechanism

Calcium-saturated calmodulin (CaM) directly activates CaM-dependent protein kinase I (CaMKI) by binding to a region in the C-terminal regulatory sequence of the enzyme to relieve autoinhibition. The structure of CaM in a high-affinity complex with a 25-residue peptide of CaMKI (residues 294-318) has been determined by X-ray crystallography at 1.7 A resolution. Upon complex formation, the CaMKI peptide adopts an alpha-helical conformation, while changes in the CaM domain linker enable both its N- and C-domains to wrap around the peptide helix. Target peptide residues Trp-303 (interacting with the CaM C-domain) and Met-316 (with the CaM N-domain) define the mode of binding as 1-14. In addition, two basic patches on the peptide form complementary charge interactions with CaM. The CaM-peptide affinity is approximately 1 pM, compared with 30 nM for the CaM-kinase complex, indicating that activation of autoinhibited CaMKI by CaM requires a costly energetic disruption of the interactions between the CaM-binding sequence and the rest of the enzyme. We present biochemical and structural evidence indicating the involvement of both CaM domains in the activation process: while the C-domain exhibits tight binding toward the regulatory sequence, the N-domain is necessary for activation. Our crystal structure also enables us to identify the full CaM-binding sequence. Residues Lys-296 and Phe-298 from the target peptide interact directly with CaM, demonstrating overlap between the autoinhibitory and CaM-binding sequences. Thus, the kinase activation mechanism involves the binding of CaM to residues associated with the inhibitory pseudosubstrate sequence.     

The neuronal voltage-dependent sodium channel type II IQ motif lowers the calcium affinity of the C-domain of calmodulin

Calmodulin (CaM) is the primary calcium sensor in eukaryotes. Calcium binds cooperatively to pairs of EF-hand motifs in each domain (N and C). This allows CaM to regulate cellular processes via calcium-dependent interactions with a variety of proteins, including ion channels. One neuronal target is NaV1.2, voltage-dependent sodium channel type II, to which CaM binds via an IQ motif within the NaV1.2 C-terminal tail (residues 1901-1938) [Mori, M., et al. (2000) Biochemistry 39, 1316-1323]. Here we report on the use of circular dichroism, fluorescein emission, and fluorescence anisotropy to study the interaction between CaM and NaV1.2 at varying calcium concentrations. At 1 mM MgCl2, both full-length CaM (CaM1-148) and a C-domain fragment (CaM76-148) exhibit tight (nanomolar) calcium-independent binding to the NaV1.2 IQ motif, whereas an N-domain fragment of CaM (CaM1-80) binds weakly, regardless of calcium concentration. Equilibrium calcium titrations of CaM at several concentrations of the NaV1.2 IQ peptide showed that the peptide reduced the calcium affinity of the CaM C-domain sites (III and IV) without affecting the N-domain sites (I and II) significantly. This leads us to propose that the CaM C-domain mediates constitutive binding to the NaV1.2 peptide, but that interaction then distorts calcium-binding sites III and IV, thereby reducing their affinity for calcium. This contrasts with the CaM-binding domains of voltage-dependent Ca2+ channels, kinases, and phosphatases, which increase the calcium binding affinity of the C-domain of CaM.     

Interdomain cooperativity of calmodulin bound to melittin preferentially increases calcium affinity of sites I and II

Calmodulin (CaM) is the primary transducer of calcium fluxes in eukaryotic cells. Its two domains allosterically regulate myriad target proteins through calcium-linked association and conformational change. Many of these proteins have a basic amphipathic alpha-helix (BAA) motif that binds one or both CaM domains. Previously, we demonstrated domain-specific binding of melittin, a model BAA peptide, to Paramecium CaM (PCaM): C-domain mutations altered the interaction with melittin, whereas N-domain mutations had no discernable effect. Here, we report on the use of fluorescence and NMR spectroscopy to measure the domain-specific association of melittin with calcium-saturated ((Ca(2+))(4)-PCaM) or calcium-depleted (apo) PCaM, which has enabled us to determine the free energies of calcium binding to the PCaM-melittin complex, and to estimate interdomain cooperativity. Under apo conditions, melittin associated with each PCaM domain fragment (PCaM(1-80) and PCaM(76-148)), as well as with the C-domain of full-length PCaM (PCaM(1-148)). In the presence of calcium, all of these interactions were again observed, in addition to which an association with the N-domain of (Ca(2+))(4)-PCaM(1-148) occurred. This new association was made possible by the fact that melittin changed the calcium-binding preferences for the domains from sequential (C > N) to concomitant, decreasing the median ligand activity of calcium toward the N-domain 10-fold more than that observed for the C-domain. This selectivity may be explained by a free energy of cooperativity of -3 kcal/mol between the N- and C-domains. This study demonstrates multiple domain-selective differences in the interactions between melittin and PCaM. Our findings support a model that may apply more generally to ion channels that associate with the C-domain of CaM under low (resting) calcium conditions, but rearrange when calcium binding triggers an association of the N- domain with the channel.     

Calcium-induced refolding of the calmodulin V136G mutant studied by NMR spectroscopy: evidence for interaction between the two globular domains

The Ca(2+) titration of the (15)N-labeled mutant V136G calmodulin has been monitored using (1)H-(15)N HSQC NMR spectra. Up to a [Ca(2+)]/[CaM] ratio of 2, the Ca(2+) ions bind predominantly to sites I and II on the N-domain in contrast with the behavior of the wild-type calmodulin where the C-terminal domain has the higher affinity for Ca(2+). Surprisingly, the Ca(2+)-binding affinity for the N-domain in the mutant calmodulin is greater than that for the N-domain in the wild-type protein. The mutated C-domain is observed as a mixture of unfolded, partially folded (site III occupied), and native-like folded (sites III and IV occupied) conformations, with relative populations dependent on the [Ca(2+)]/[CaM] ratio. The occupancy of site III independently of site IV in this mutant shows that the cooperativity of Ca(2+) binding in the C-domain is mediated by the integrity of the domain structure. Several NH signals from residues in the Ca(2+)-bound N-domain appear as two signals during the Ca(2+) titration indicating separate species in slow exchange, and it can be deduced that these result from the presence and absence of interdomain interactions in the mutant. It is proposed that an unfolded part of the mutated C-domain interacts with sites on the N-domain that normally bind to target proteins. This would also account for the increase in the Ca(2+) affinity for the N-domain in the mutant compared with the wild-type calmodulin. The results therefore show the wide-ranging effects of a point mutation in a single Ca(2+)-binding site, providing details of the involvement of individual residues in the calcium-induced folding reactions.     

Calcium binding to calmodulin mutants having domain-specific effects on the regulation of ion channels

Calmodulin (CaM) is an essential, eukaryotic protein comprised of two highly homologous domains (N and C). CaM binds four calcium ions cooperatively, regulating a wide array of target proteins. A genetic screen of Paramecia by Kung [Kung, C. et al. (1992) Cell Calcium 13, 413-425] demonstrated that the domains of CaM have separable physiological roles: "under-reactive" mutations affecting calcium-dependent sodium currents mapped to the N-domain, while "over-reactive" mutations affecting calcium-dependent potassium currents localized to the C-domain of CaM. To determine whether and how these mutations affected intrinsic calcium-binding properties of CaM domains, phenylalanine fluorescence was used to monitor calcium binding to sites I and II (N-domain) and tyrosine fluorescence was used to monitor sites III and IV (C-domain). To explore interdomain interactions, binding properties of each full-length mutant were compared to those of its corresponding domain fragments. The calcium-binding properties of six under-reactive mutants (V35I/D50N, G40E, G40E/D50N, D50G, E54K, and G59S) and one over-reactive mutant (M145V) were indistinguishable from those of wild-type CaM, despite their deleterious physiological effects on ion-channel regulation. Four over-reactive mutants (D95G, S101F, E104K, and H135R) significantly decreased the calcium affinity of the C-domain. Of these, one (E104K) also increased the calcium affinity of the N-domain, demonstrating that the magnitude and direction of wild-type interdomain coupling had been perturbed. This suggests that, while some of these mutations alter calcium-binding directly, others probably alter CaM-channel association or calcium-triggered conformational change in the context of a ternary complex with the affected ion channel.     

Paramecium calmodulin mutants defective in ion channel regulation associate with melittin in the absence of calcium but require it for tertiary collapse

Calmodulin (CaM) is a small acidic protein essential to calcium-mediated signal transduction. Conformational change driven by calcium binding controls its selective activation of myriad target proteins. In most well characterized cases, both homologous domains of CaM interact with a target protein. However, physiologically separable roles for the two domains were demonstrated by mutants of Paramecium tetraurelia [Kung, C. et al. (1992) Cell Calcium 13, 413], some of which have altered calcium affinities [Jaren, O. R. et al. (2000) Biochemistry 39, 6881]. To determine whether these mutants can associate with canonical targets in a calcium-dependent manner, their ability to bind melittin was assessed using analytical gel permeation chromatography, analytical ultracentrifugation, and fluorescence spectroscopy. The Stokes radius of wild-type PCaM and 11 of the mutants decreased dramatically upon binding melittin in the presence of calcium. Fluorescence spectra and sedimentation velocity studies showed that melittin bound to wild-type PCaM and mutants in a calcium-independent manner. However, there were domain-specific perturbations. Mutations in the N-domain of PCaM did not affect the spectrum of melittin (residue W19) under apo or calcium-saturated conditions, whereas most of the mutations in the C-domain did. These data are consistent with a calcium-dependent model of sequential target association whereby melittin (i) binds to the C-domain of PCaM in the absence of calcium, (ii) remains associated with the C-domain upon calcium binding to sites III and IV, and (iii) subsequently binds to the N-domain upon calcium binding to sites I and II of CaM, causing tertiary collapse.     

Target recognition by calmodulin: dissecting the kinetics and affinity of interaction using short peptide sequences.

The interaction between calmodulin (CaM) and peptide M13, its target binding sequence from skeletal muscle myosin light chain kinase, involves predominantly two sets of interactions, between the N-terminal target residues and the C-domain of calmodulin, and between the C-terminal target residues and the N-domain of calmodulin (Ikura M et al., 1992, Science 256:632-638). Using short synthetic peptides based on the two halves of the target sequence, the interactions with calmodulin and its separate C-domain have been studied by fluorescence and CD spectroscopy, calcium binding, and kinetic techniques. Peptide WF10 (residues 1-10 of M13) binds to CaM with Kd approximately 1 microM; peptide FW10 (residues 9-18 of M13, with Phe-17-->Trp substitution) binds to CaM with Kd approximately 100 microM. The effect of peptide WF10 on calcium binding to calmodulin produces a biphasic saturation curve, with marked enhancement of affinity for the binding of two calcium ions to the C-domain, forming a stable half-saturated complex, Ca2-CaM-peptide, and confirming the functional importance of the interaction of this sequence with the C-domain. Stopped-flow studies show that the EGTA-induced dissociation of WF10 from Ca4-CaM proceeds by a reversible relaxation mechanism from a kinetic intermediate state, also involving half-saturation of CaM, and the same mechanism is evident for the full target peptide. Interaction of the N-terminal target residues with the C-domain is energetically the most important component, but interaction of calmodulin with the whole target sequence is necessary to induce the full cooperative interaction of the two contiguous elements of the target sequence with both N- and C-domains of calmodulin. Thus, the interaction of calmodulin with the M13 sequence can be dissected on both a structural and kinetic basis into partial reactions involving intermediates comprising distinct regions of the target sequence. We propose a general mechanism for the calcium regulation of calmodulin-dependent enzyme activation, involving an intermediate complex formed by interaction of the calmodulin C-domain and the corresponding part of the target sequence. This intermediate species can function to regulate the overall calcium sensitivity of activation and to determine the affinity of the calmodulin target interaction.     

A model for the calmodulin-peptide complex based on the troponin C crystal packing and its similarity to the NMR structure of the calmodulin-myosin light chain kinase peptide complex.

In the crystal structure of troponin C, the holo C-domain is bound in a head-to-tail fashion to the A-helix of the apo N-domain of a symmetry-related molecule. Using this interaction, we have proposed a model for the calmodulin-peptide complex. We find that the interaction of the C-domain with the A-helix is similar to that observed in the NMR structure of the calmodulin-myosin light chain kinase (MLCK) peptide complex. This similarity in binding has enabled us to make a precise sequence alignment of the target peptides in the calmodulin-binding cleft and to rationalize the amino acid sequence-dependent binding strengths of various peptides. Our model differs from that proposed by Strynadka and James (Proteins Struct. Funct. Genet. 7, 234-248, 1990) in that the peptides are rotated by 100 degrees in the calmodulin binding cleft.     

Static and kinetic studies of complex formations between calmodulin and mastoparanX

Ca(2+)-induced complex formation between calmodulin (CaM) and mastoparanX (MasX) was studied by a fluorescence spectroscopy and by a stopped-flow method. The measurements of the fluorescence anisotropy in the presence of calcium and the fluorescence titration with Ca(2+) revealed that the N- and C-domains of CaM bound cooperatively MasX, while the tryptic fragments of CaM (TR(1)C, 1-77 and TR(2)C, 78-148) bound independently MasX. The Trp-fluorescence stopped-flow experiments revealed that the Ca(2+)-induced binding of CaM and MasX was composed of two processes: one was a rapid binding of the N-domain of CaM to MasX, which was induced by the rapid Ca(2+) binding to the N-sites of CaM. The other was a slow biphasic process. Its fast phase was the binding of the C-domain of CaM to MasX, which was induced by the slow Ca(2+) binding to the C-sites. Interestingly, the kinetics of the slow process varied with the Ca(2+) concentrations. At the low Ca(2+) concentrations, its rate constant increased to around 20 s(-1) as the Ca(2+) concentration increased. At the high Ca(2+) concentrations, the Ca(2+)-induced binding of the C-domain of CaM to MasX proceeded at a constant rate around 20 s(-1). This suggested an existence of a rate-limiting step for the Ca(2+)-induced binding of the C-domain of CaM to MasX at the high Ca(2+) concentrations. The slow phase of the slow process may be a rearrangement of the CaM-MasX complex. These results led to our model of a molecular kinetic mechanism of the Ca(2+)-induced complex formation between CaM and MasX.     

A molecular switch in SecA protein couples ATP hydrolysis to protein translocation

SecA, the dimeric ATPase subunit of bacterial protein translocase, catalyses translocation during ATP-driven membrane cycling at SecYEG. We now show that the SecA protomer comprises two structural modules: the ATPase N-domain, containing the nucleotide binding sites NBD1 and NBD2, and the regulatory C-domain. The C-domain binds to the N-domain in each protomer and to the C-domain of another protomer to form SecA dimers. NBD1 is sufficient for single rounds of SecA ATP hydrolysis. Multiple ATP turnovers at NBD1 require both the NBD2 site acting in cis and a conserved C-domain sequence operating in trans. This intramolecular regulator of ATP hydrolysis (IRA) mediates N-/C-domain binding and acts as a molecular switch: it suppresses ATP hydrolysis in cytoplasmic SecA while it releases hydrolysis in SecY-bound SecA during translocation. We propose that the IRA switch couples ATP binding and hydrolysis to SecA membrane insertion/deinsertion and substrate translocation by controlling nucleotide-regulated relative motions between the N-domain and the C-domain. The IRA switch is a novel essential component of the protein translocation catalytic pathway.     

Direct monitoring of allosteric recognition of type IIE restriction endonuclease EcoRII

EcoRII is a homodimer with two domains consisting of a DNA-binding N terminus and a catalytic C terminus and recognizes two specific sequences on DNA. It shows a relatively complicated cleavage reaction in bulk solution. After binding to either recognition site, EcoRII cleaves the other recognition site of the same DNA (cis-binding) strand and/or the recognition site of the other DNA (trans-binding) strand. Although it is difficult to separate these two reactions in bulk solution, we could simply obtain the binding and cleavage kinetics of only the cis-binding by following the frequency (mass) changes of a DNA-immobilized quartz-crystal microbalance (QCM) responding to the addition of EcoRII in aqueous solution. We obtained the maximum binding amounts (Deltam(max)), the dissociation constants (K(d)), the binding and dissociation rate constants (k(on) and k(off)), and the catalytic cleavage reaction rate constants (k(cat)) for wild-type EcoRII, the N-terminal-truncated form (EcoRII N-domain), and the mutant derivatives in its C-terminal domain (K263A and R330A). It was determined from the kinetic analyses that the N-domain, which covers the catalytic C-domain in the absence of DNA, preferentially binds to the one DNA recognition site while transforming EcoRII into an active form allosterically, and then the secondary C-domain binds to and cleaves the other recognition site of the DNA strand.     

Conformation of the calmodulin-binding domain of metabotropic glutamate receptor subtype 7 and its interaction with calmodulin

Calmodulin (CaM), a Ca(2+)-binding protein, is a well-known regulator of various cellular functions. One of the targets of CaM is metabotropic glutamate receptor 7 (mGluR7), which serves as a low-pass filter for glutamate in the pre-synaptic terminal to regulate neurotransmission. Surface plasmon resonance (SPR), circular dichroism (CD) spectroscopy and nuclear magnetic spectroscopy (NMR) were performed to study the structure of the peptides corresponding to the CaM-binding domain of mGluR7 and their interaction with CaM. Unlike well-known CaM-binding peptides, mGluR7 has a random coil structure even in the presence of trifluoroethanol. Moreover, NMR data suggested that the complex between Ca(2+)/CaM and the mGluR7 peptide has multiple conformations. The mGluR7 peptide has been found to interact with CaM even in the absence of Ca(2+), and the binding is directed toward the C-domain of apo-CaM rather than the N-domain. We propose a possible mechanism for the activation of mGluR7 by CaM. A pre-binding occurs between apo-CaM and mGluR7 in the resting state of cells. Then, the Ca(2+)/CaM-mGluR7 complex is formed once Ca(2+) influx occurs. The weak interaction at lower Ca(2+) concentrations is likely to bind CaM to mGluR7 for the fast complex formation in response to the elevation of Ca(2+) concentration.     

Calcium binding to calmodulin mutants monitored by domain-specific intrinsic phenylalanine and tyrosine fluorescence

Cooperative calcium binding to the two homologous domains of calmodulin (CaM) induces conformational changes that regulate its association with and activation of numerous cellular target proteins. Calcium binding to the pair of high-affinity sites (III and IV in the C-domain) can be monitored by observing calcium-dependent changes in intrinsic tyrosine fluorescence intensity (lambda(ex)/lambda(em) of 277/320 nm). However, calcium binding to the low-affinity sites (I and II in the N-domain) is more difficult to measure with optical spectroscopy because that domain of CaM does not contain tryptophan or tyrosine. We recently demonstrated that calcium-dependent changes in intrinsic phenylalanine fluorescence (lambda(ex)/lambda(em) of 250/280 nm) of an N-domain fragment of CaM reflect occupancy of sites I and II (VanScyoc, W. S., and M. A. Shea, 2001, Protein Sci. 10:1758-1768). Using steady-state and time-resolved fluorescence methods, we now show that these excitation and emission wavelength pairs for phenylalanine and tyrosine fluorescence can be used to monitor equilibrium calcium titrations of the individual domains in full-length CaM. Calcium-dependent changes in phenylalanine fluorescence specifically indicate ion occupancy of sites I and II in the N-domain because phenylalanine residues in the C-domain are nonemissive. Tyrosine emission from the C-domain does not interfere with phenylalanine fluorescence signals from the N-domain. This is the first demonstration that intrinsic fluorescence may be used to monitor calcium binding to each domain of CaM. In this way, we also evaluated how mutations of two residues (Arg74 and Arg90) located between sites II and III can alter the calcium-binding properties of each of the domains. The mutation R74A caused an increase in the calcium affinity of sites I and II in the N-domain. The mutation R90A caused an increase in calcium affinity of sites III and IV in the C-domain whereas R90G caused an increase in calcium affinity of sites in both domains. This approach holds promise for exploring the linked energetics of calcium binding and target recognition.     

Structure and characterization of RNase H3 from Aquifex aeolicus

The crystal structure of ribonuclease?H3 from Aquifex?aeolicus (Aae-RNase?H3) was determined at 2.0?? resolution. Aae-RNase?H3 consists of an N-terminal TATA box-binding protein (TBP)-like domain (N-domain) and a C-terminal RNase?H domain (C-domain). The structure of the C-domain highly resembles that of Bacillus?stearothermophilus RNase?H3 (Bst-RNase?H3), except that it contains three disulfide bonds, and the fourth conserved glutamate residue of the Asp-Glu-Asp-Glu active site motif (Glu198) is located far from the active site. These disulfide bonds were shown to contribute to hyper-stabilization of the protein. Non-conserved Glu194 was identified as the fourth active site residue. The structure of the N-domain without the C-domain also highly resembles that of Bst-RNase?H3. However, the arrangement of the N-domain relative to the C-domain greatly varies for these proteins because of the difference in the linker size between the domains. The linker of Bst-RNase?H3 is relatively long and flexible, while that of Aae-RNase?H3 is short and assumes a helix formation. Biochemical characterizations of Aae-RNase?H3 and its derivatives without the N- or C-domain or with a mutation in the N-domain indicate that the N-domain of Aae-RNase?H3 is important for substrate binding, and uses the flat surface of the β-sheet for substrate binding. However, this surface is located far from the active site and on the opposite side to the active site. We propose that the N-domain of Aae-RNase?H3 is required for initial contact with the substrate. The resulting complex may be rearranged such that only the C-domain forms a complex with the substrate.     

Differential binding of calmodulin domains to constitutive and inducible nitric oxide synthase enzymes

Calmodulin (CaM) is a Ca2+ signal transducing protein that binds and activates many cellular enzymes with physiological relevance, including the mammalian nitric oxide synthase (NOS) isozymes: endothelial NOS (eNOS), neuronal NOS (nNOS), and inducible NOS (iNOS). The mechanism of CaM binding and activation to the iNOS enzyme is poorly understood in part due to the strength of the bound complex and the difficulty of assessing the role played by regions outside of the CaM-binding domain. To further elucidate these processes, we have developed the methodology to investigate CaM binding to the iNOS holoenzyme and generate CaM mutant proteins selectively labeled with fluorescent dyes at specific residues in the N-terminal lobe, C-terminal lobe, or linker region of the protein. In the present study, an iNOS CaM coexpression system allowed for the investigation of CaM binding to the holoenzyme; three different mutant CaM proteins with cysteine substitutions at residues T34 (N-domain), K75 (central linker), and T110 (C-domain) were fluorescently labeled with acrylodan or Alexa Fluor 546 C5-maleimide. These proteins were used to investigate the differential association of each region of CaM with the three NOS isoforms. We have also N-terminally labeled an iNOS CaM-binding domain peptide with dabsyl chloride in order to perform FRET studies between Alexa-labeled residues in the N- and C-terminal domains of CaM to determine CaM's orientation when associated to iNOS. Our FRET results show that CaM binds to the iNOS CaM-binding domain in an antiparallel orientation. Our steady-state fluorescence and circular dichroism studies show that both the N- and C-terminal EF hand pairs of CaM bind to the CaM-binding domain peptide of iNOS in a Ca2+-independent manner; however, only the C-terminal domain showed large Ca2+-dependent conformational changes when associated with the target sequence. Steady-state fluorescence showed that Alexa-labeled CaM proteins are capable of binding to holo-iNOS coexpressed with nCaM, but this complex is a transient species and can be displaced with the addition of excess CaM. Our results show that CaM does not bind to iNOS in a sequential manner as previously proposed for the nNOS enzyme. This investigation provides additional insight into why iNOS remains active even under basal levels of Ca2+ in the cell.     

自旋标记钙调蛋白与酸枣仁皂甙A相互作用研究

中草药有效成份酸枣仁皂甙A是钙调蛋白CaM的一种天然非竞争性拮抗剂。利用氮氧自由基马来酰亚胺衍生物标记CaM研究了二者的相互作用。结果表明,每分子CaM至少可以结合二个分子的酸枣仁皂钙A,二者的作用影响CaM上Lys残基主要是75,94)的环境,推测酸枣仁皂钙A是通过疏水作用结合到CaM分子两端的疏水沟区。通过比较三氟拉嗪TFP与酸枣仁皂甙A的结构特点,抑制性质与结合位点,提出了CaM调节环核苷酸磷酸二酯酶PDE的模式。     

Selectivity of stop codon recognition in translation termination is modulated by multiple conformations of GTS loop in eRF1

Translation termination in eukaryotes is catalyzed by two release factors eRF1 and eRF3 in a cooperative manner. The precise mechanism of stop codon discrimination by eRF1 remains obscure, hindering drug development targeting aberrations at translation termination. By solving the solution structures of the wild-type N-domain of human eRF1 exhibited omnipotent specificity, i.e. recognition of all three stop codons, and its unipotent mutant with UGA-only specificity, we found the conserved GTS loop adopting alternate conformations. We propose that structural variability in the GTS loop may underline the switching between omnipotency and unipotency of eRF1, implying the direct access of the GTS loop to the stop codon. To explore such feasibility, we positioned N-domain in a pre-termination ribosomal complex using the binding interface between N-domain and model RNA oligonucleotides mimicking Helix 44 of 18S rRNA. NMR analysis revealed that those duplex RNA containing 2-nt internal loops interact specifically with helix α1 of N-domain, and displace C-domain from a non-covalent complex of N-domain and C-domain, suggesting domain rearrangement in eRF1 that accompanies N-domain accommodation into the ribosomal A site.