Sulphydryl groups of (Na+ + K+)-ATPase from rectal glands of Squalus acanthias: Detection of ligand-induced conformational changes

1. Modification of the Class II sulphydryl groups on the (Na⁺ + K⁺)-ATPase from rectal glands of Squalus acanthias with N-ethylmaleimide has been used to detect conformational changes in the protein. The rates of inactivation of the enzyme and the incorporation of N-ethylmaleimide depend on the ligands present in the incubation medium. With 150 mM K⁺ the rate of inactivation is largest (k₁ = 1.73 mM^?1 · min^?1) and four SH groups per α-subunit are modified. The rate of inactivation in the presence of 150 mM Na⁺ is smaller (k₁ = 1.08 mM^?1 · min^-1) but the incorporation of N-ethylmaleimide is the same as with K⁺. 2. ATP in micromolar concentrations protects the Class II groups in the presence of Na⁺ (k₁ = 0.08 mM^?1 · min^?1 at saturating ATP) and the incorporation id drastically reduced. ATP in millimolar concentrations protects the Class II groups partially in the presence of K⁺ (k₁ = 1.08 mM^?1 · min^?1) and three SH groups are labelled per α subunit. 3. The K⁺ -dependent phosphatase is inhibited in parallel to the (Na⁺ + K⁺)-ATPase under all conditions, and the ligand-dependent incorporation of N-ethylmaleimide was on the α-subunit only. 4. It is shown that the difference between the Na⁺ and K⁺ conformations sensed with N-ethylmaleimide depends on the pH of the incubation medium. At pH 6 there is a very small difference between the rates of inactivation in the presence of Na⁺ and K⁺, but at higher pH the difference increases. It is also shown that the rate of inactivation has a minimum at pH 6.9, which suggests that the conformation of the enzyme changes with pH. 5. Modification of the Class III groups with N-ethylmaleimide-whereby the enzyme activity is reduced from about 16% to zero-shows that these groups are also sensitive to conformational changes. As with the Class II groups, ATP in micromolar concentrations protects in the presence of Na⁺ relative to Na⁺ or K⁺ alone. ATP in millimolar concentrations with K⁺ present increases the rate of inactivation relative to K⁺ alone, in contrast to the effect on the Class II groups. 6. Modification of the Class II groups with a maleimide spin label shows a difference between Class II groups labelled in the presence of Na⁺ (or K⁺) and Class II groups labelled in the presence of K + ATP, in agreement with the difference in incorporation of N-ethylmaleimide. The spectra suggest that the SH group protected by ATP in the presence of K⁺ is buried in the protein. 7. The results suggest that at least four different conformations of the (Na⁺ + K⁺)-ATPase can be sensed with N-ethylmaleimide: (i) a Na⁺ form of the enzyme with ATP bound to a high-affinity site (E₁-Na-ATP); (ii) a Na⁺ form without ATP bound (E₁-Na); (iii) a K⁺ form without ATP bound (E₂-K); and (iv) an enzyme form with ATP bound to a low-affinity site in the presence of K⁺, probably and E₁-K-ATP form.     

锌离子对氨基酰化酶构象及其稳定性的影响

天然氨基酰化酶和脱谷氨基酰化酶无论在二级结构（用ＣＤ和ＦＴＩＲ监测）还是三级结构上（以荧光发射光谱监测）都有明显的差异,表明了脱锌后酶的有序度降低;当比较天然和脱锌氨基酸化酶对去圬剂的稳定性时,结果表明脱锌后酶的构象的稳定性明显降低．因此可以认为锌离子对维持酶分子活性部位的特定构象以及构象的稳定性具有重要的作用．     

The CC' and DE loops in Ig domains 1 and 2 of MAdCAM-1 play different roles in MAdCAM-1 binding to low- and high-affinity integrin alpha4beta7

Lymphocyte homing is regulated by the dynamic interaction between integrins and their ligands. Integrin α4β7 mediates both rolling and firm adhesion of lymphocytes by modulating its affinity to the ligand, mucosal addressin cell adhesion molecule-1 (MAdCAM-1). Although previous studies have revealed some mechanisms of α4β7-MAdCAM-1 binding, little is known about the different molecular bases of the low- and high-affinity α4β7-MAdCAM-1 interactions, which mediate rolling and firm adhesion of lymphocytes, respectively. Here, we found that two loops in immunoglobulin domains 1 and 2 (D1 and D2) of MAdCAM-1 played different roles in MAdCAM-1 binding to low-affinity (inactive) and high-affinity (activated) α4β7. The Asp-42 in the CC' loop of D1 was indispensable for MAdCAM-1 binding to both low-affinity and high-affinity α4β7. The other CC' loop residues except for Arg-39 and Ser-44 were essential for MAdCAM-1 binding to both inactive α4β7 and α4β7 activated by SDF-1α or talin, but not required for MAdCAM-1 binding to Mn2+-activated α4β7. Single amino acid substitution of the DE loop residues mildly decreased MAdCAM-1 binding to both inactive and activated α4β7. Notably, removal of the DE loop greatly impaired MAdCAM-1 binding to inactive and SDF-1α- or talin-activated α4β7, but only decreased 60% of MAdCAM-1 binding to Mn2+-activated α4β7. Moreover, DE loop residues were important for stabilizing the low-affinity α4β7-MAdCAM-1 interaction. Thus, our findings demonstrate the distinct roles of the CC' and DE loops in the recognition of MAdCAM-1 by low- and high-affinity α4β7 and suggest that the inactive α4β7 and α4β7 activated by different stimuli have distinct conformations with different structural requirements for MAdCAM-1 binding.     

Synthetic Dimeric Aβ(28–40) Mimics the Complex Epitope of Human Anti-Aβ Autoantibodies against Toxic Aβ Oligomers

Covalently linked carboxyl-terminal segments of the β-amyloid peptide (Aβ) were tested for their qualification as minimal conformational epitopes of the naturally occurring human autoantibodies against β-amyloid (nAbs-Aβ). nAbs-Aβ specifically recognize the toxic oligomers of Aβ and not the monomeric or the fibrillar forms of Aβ. The synthetic dimers of Aβ(28–40) described herein mimic the toxic Aβ oligomers but are not kinetic intermediates with uncertain compositions. CD spectra identified a surprisingly rich conformational behavior of selected miniamyloids. We observed a highly cooperative conformational transition of β-sheet to α-helix upon the addition of the helix enforcing co-solvent hexafluoroisopropanol. The CD curves of dimer 9 resembled, in a completely reversible manner, the CD spectra measured during the irreversible fibrillation of the parent Aβ(1–40). Synthetic peptide epitopes with high affinities for nAbs-Aβ are needed to identify the physiological roles of nAbs-Aβ and are promising epitopes for vaccination experiments.     

人肌肌酸激酶胍变性时的失活与构象变化的比较研究

应用二阶导数光谱、紫外差吸收光谱和荧光光谱等监测手段,研究了人肌肌酸激酶在盐酸胍溶液中的构象变化。二阶导数光谱结果表明,若以6M盐酸胍中肌酸激酶酪氨酸残基的暴露程度为100％,则天然酶酪氨酸残基的暴露程度只有2％。而紫外差吸收光谱和荧光光谱的变化与兔肌肌酸激酶的结果相似。比较不同胍浓度下人肌肌酸激酶的失活与构象变化,表明酶的失活先于构象变化。同时还测定了不同浓度胍溶液中人肌酶的失活与构象变化的速度常数。结果表明以几种方法测定的构象变化均为单相的一级过程,而酶的失活却呈现了由快慢两相组成的一级反应过程。比较同浓度胍溶液中的失活速度与构象变化速度,发现酶失活的快相反应速度常数比构象变化的速度常数大1—2个数量级,慢相速度常数与构象变化速度常数相近。上述结果进一步支持了酶的活性部位构象柔性的观点。     

Substrate Binding Stabilizes a Pre-translocation Intermediate in the ATP-binding Cassette Transport Protein MsbA

ATP-binding cassette (ABC) transporters belong to one of the largest protein superfamilies that expands from prokaryotes to man. Recent x-ray crystal structures of bacterial and mammalian ABC exporters suggest a common alternating access mechanism of substrate transport, which has also been biochemically substantiated. However, the current model does not yet explain the coupling between substrate binding and ATP hydrolysis that underlies ATP-dependent substrate transport. In our studies on the homodimeric multidrug/lipid A ABC exporter MsbA from Escherichia coli, we performed cysteine cross-linking, fluorescence energy transfer, and cysteine accessibility studies on two reporter positions, near the nucleotide-binding domains and in the membrane domains, for transporter embedded in a biological membrane. Our results suggest for the first time that substrate binding by MsbA stimulates the maximum rate of ATP hydrolysis by facilitating the dimerization of nucleotide-binding domains in a state, which is markedly distinct from the previously described nucleotide-free, inward-facing and nucleotide-bound, outward-facing conformations of ABC exporters and which binds ATP.     

The mechanism of fibril formation of a non-inhibitory serpin ovalbumin revealed by the identification of amyloidogenic core regions

Ovalbumin (OVA), a non-inhibitory member of the serpin superfamily, forms fibrillar aggregates upon heat-induced denaturation. Recent studies suggested that OVA fibrils are generated by a mechanism similar to that of amyloid fibril formation, which is distinct from polymerization mechanisms proposed for other serpins. In this study, we provide new insights into the mechanism of OVA fibril formation through identification of amyloidogenic core regions using synthetic peptide fragments, site-directed mutagenesis, and limited proteolysis. OVA possesses a single disulfide bond between Cys(73) and Cys(120) in the N-terminal helical region of the protein. Heat treatment of disulfide-reduced OVA resulted in the formation of long straight fibrils that are distinct from the semiflexible fibrils formed from OVA with an intact disulfide. Computer predictions suggest that helix B (hB) of the N-terminal region, strand 3A, and strands 4-5B are highly β-aggregation-prone regions. These predictions were confirmed by the fact that synthetic peptides corresponding to these regions formed amyloid fibrils. Site-directed mutagenesis of OVA indicated that V41A substitution in hB interfered with the formation of fibrils. Co-incubation of a soluble peptide fragment of hB with the disulfide-intact full-length OVA consistently promoted formation of long straight fibrils. In addition, the N-terminal helical region of the heat-induced fibril of OVA was protected from limited proteolysis. These results indicate that the heat-induced fibril formation of OVA occurs by a mechanism involving transformation of the N-terminal helical region of the protein to β-strands, thereby forming sequential intermolecular linkages.     

Conformational Changes Produced by ATP Binding to the Plasma Membrane Calcium Pump

The aim of this work was to study the plasma membrane calcium pump (PMCA) reaction cycle by characterizing conformational changes associated with calcium, ATP, and vanadate binding to purified PMCA. This was accomplished by studying the exposure of PMCA to surrounding phospholipids by measuring the incorporation of the photoactivatable phosphatidylcholine analog 1-O-hexadecanoyl-2-O-[9-[[[2-[¹²⁵I]iodo-4-(trifluoromethyl-3H-diazirin-3-yl)benzyl]oxy]carbonyl]nonanoyl]-sn-glycero-3-phosphocholine to the protein. ATP could bind to the different vanadate-bound states of the enzyme either in the presence or in the absence of Ca²⁺ with high apparent affinity. Conformational movements of the ATP binding domain were determined using the fluorescent analog 2′(3′)-O-(2,4,6-trinitrophenyl)adenosine 5′-triphosphate. To assess the conformational behavior of the Ca²⁺ binding domain, we also studied the occlusion of Ca²⁺, both in the presence and in the absence of ATP and with or without vanadate. Results show the existence of occluded species in the presence of vanadate and/or ATP. This allowed the development of a model that describes the transport of Ca²⁺ and its relation with ATP hydrolysis. This is the first approach that uses a conformational study to describe the PMCA P-type ATPase reaction cycle, adding important features to the classical E₁-E₂ model devised using kinetics methodology only.     

Cdc37 (Cell Division Cycle 37) Restricts Hsp90 (Heat Shock Protein 90) Motility by Interaction with N-terminal and Middle Domain Binding Sites

The ATPase-driven dimeric molecular Hsp90 (heat shock protein 90) and its cofactor Cdc37 (cell division cycle 37 protein) are crucial to prevent the cellular depletion of many protein kinases. In complex with Hsp90, Cdc37 is thought to bind an important lid structure in the ATPase domain of Hsp90 and inhibit ATP turnover by Hsp90. As different interaction modes have been reported, we were interested in the interaction mechanism of Hsp90 and Cdc37. We find that Cdc37 can bind to one subunit of the Hsp90 dimer. The inhibition of the ATPase activity is caused by a reduction in the closing rate of Hsp90 without obviously bridging the two subunits or affecting nucleotide accessibility to the binding site. Although human Cdc37 binds to the N-terminal domain of Hsp90, nematodal Cdc37 preferentially interacts with the middle domain of CeHsp90 and hHsp90, exposing two Cdc37 interaction sites. A previously unreported site in CeCdc37 is utilized for the middle domain interaction. Dephosphorylation of CeCdc37 by the Hsp90-associated phosphatase PPH-5, a step required during the kinase activation process, proceeds normally, even if only the new interaction site is used. This shows that the second interaction site is also functionally relevant and highlights that Cdc37, similar to the Hsp90 cofactors Sti1 and Aha1, may utilize two different attachment sites to restrict the conformational freedom and the ATP turnover of Hsp90.