Mapping Allostery through Equilibrium Perturbation NMR Spectroscopy

Das, Rahul; Abu-Abed, Mona; Melacini, Giuseppe

doi:10.1021/ja060046d

Cited by 34 publications

(44 citation statements)

References 25 publications

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“…2, A and B), it is likely that the associated RI␣ segments comprising PBC residues and the ␣C helices are unstructured or ill structured in the apo-state, exhibiting conformational averaging on an intermediate chemical shift (micro-to millisecond) time scale. This is in agreement with previous NMR studies of the apo form of the isolated A domain that reported a destabilization of the PBC region, evidenced by increased solvent exposure and dynamics upon the removal of cAMP (11,12). Interestingly, all regions associated with high conformational flexibility in the apo-form are localized within the primary recognition area for the C subunit (4).…”

Section: Ri␣-(98 -381) Monomer Binds Camp and Camp Analogssupporting

confidence: 93%

“…However, little is known about the structure of the ligand-free (apo) state of the R subunits. A truncated RI␣ (residues 119 -244), comprising most of the A domain, has been investigated by NMR (11)(12)(13). Note, however, that this truncated form lacks not only the B domain but also the C-terminal end of the A domain, in particular the ␣C:A and ␣CЈ:A helices.…”

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confidence: 99%

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Allosteric Communication between cAMP Binding Sites in the RI Subunit of Protein Kinase A Revealed by NMR

Byeon

Dao

Jung

et al. 2010

Journal of Biological Chemistry

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The activation of protein kinase A involves the synergistic binding of cAMP to two cAMP binding sites on the inhibitory R subunit, causing release of the C subunit, which subsequently can carry out catalysis. We used NMR to structurally characterize in solution the RI␣-(98 -381) subunit, a construct comprising both cyclic nucleotide binding (CNB) domains, in the presence and absence of cAMP, and map the effects of cAMP binding at single residue resolution. Several conformationally disordered regions in free RI␣ become structured upon cAMP binding, including the interdomain ␣C:A and ␣C:A helices that connect CNB domains A and B and are primary recognition sites for the C subunit. NMR titration experiments with cAMP, B site-selective 2-Cl-8-hexylamino-cAMP, and A site-selective Protein kinase A (PKA)3 is a primary receptor for cyclic adenosine monophosphate (cAMP) in eukaryotic cells (1, 2). In the absence of cAMP, the enzyme is an inactive, tetrameric holoenzyme complex, composed of two regulatory (R) and two catalytic (C) subunits (R 2 C 2 ). The catalytic site of the C subunit is occluded by a short inhibitory sequence in the R subunit (residues 94 -99 in bovine RI␣) that connects the N-terminal dimerization domain to the two cyclic nucleotide binding (CNB) domains. Multiple contacts exist between the CNB domains and the C subunit. The enzyme is allosterically activated by cAMP (3, 4), whose binding to the R subunits causes dissociation of the C subunits from the holoenzyme complex, thereby rendering C catalytically active (5). Two CNB domains (A and B) are present in all four isoforms (RI␣, RI␤, RII␣, and RII␤) of mammalian PKA, and both need to be occupied by cAMP to achieve PKA dissociation under physiologically relevant conditions (for reviews, see Refs. 2 and 6). Newly transcribed R subunit (apoR) in the cell can complex with either cAMP or the C subunit of PKA. Binding of cAMP leads to a dramatically decreased affinity for the C subunit, whereas binding of the C subunit lowers the cAMP affinity by about 3 orders of magnitude (7), allowing the holoenzyme to respond to fluctuations in physiological cAMP concentrations (8, 9).Comparing the crystal structures of RI␣-(103-376) (numbering for bovine RI␣) with cAMP bound in both the A and B domains (10) with the structure of RI␣-(91-379) (R333K) complexed with the C subunit (4) revealed pronounced differences in the two CNB domains, in particular with respect to their relative positioning (Fig. 1). However, little is known about the structure of the ligand-free (apo) state of the R subunits. A truncated RI␣ (residues 119 -244), comprising most of the A domain, has been investigated by NMR (11-13). Note, however, that this truncated form lacks not only the B domain but also the C-terminal end of the A domain, in particular the ␣C:A and ␣CЈ:A helices. These helices are at the junction between domains A and B and are important elements for interaction with the C subunit (4). Moreover, this region is conserved in all CAP-related eukaryotic cAMP binding proteins, as...

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Section: Ri␣-(98 -381) Monomer Binds Camp and Camp Analogssupporting

confidence: 93%

mentioning

confidence: 99%

Allosteric Communication between cAMP Binding Sites in the RI Subunit of Protein Kinase A Revealed by NMR

Byeon

Dao

Jung

et al. 2010

Journal of Biological Chemistry

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“…Peaks affected by partial or total overlap were excluded from analysis. The NMR relaxation dispersion (⌬R 2,eff ) was quantified as (32) as shown in Equations 2-4, (26), the dihedral angle restraints predicted from NMR chemical shifts using TALOS (35), hydrogen bonding restraints from the Chemical Shift Index (36), NOEs (NH-NH, NH-methyl, and methyl-methyl), and metal ion binding coordinates (15). For the NH-NH and NH-methyl NOEs from the 15 N-edited HSQC-NOESY experiment (mixing time 120 ms), two categories (1.8 -5.0 and 1.8 -6.0 Å) were created based on the peak intensities; for the methyl-methyl NOEs from 13 C-HMQC-NOESY (mixing time 200 ms) acquired with a sample in D 2 O solvent, two slightly larger categories (1.8 -6.0 and 1.8 -8.0 Å) were implemented.…”

Section: Methodsmentioning

confidence: 99%

“…It has been widely accepted that slow motion dynamics (microsecond to millisecond time scale) is directly relevant to the biological function of many proteins (20,49,50). In particular, solution dynamics CPMG relaxation dispersion measurements have been used to characterize protein conformational exchange (21), ligandbinding site chemical exchange (51), and allostery mapping (32). In our studies, the CPMG slow motion dynamics experiments for the backbone residues of Ca 2ϩ -CIB1, Mg 2ϩ -CIB1, as well as Ca 2ϩ -CIB1 and Mg 2ϩ -CIB1 in complex with the ␣IIb peptide were carried out.…”

Section: Methyl Groups Probe the Interaction Between Cib1 And ␣Iib-mentioning

confidence: 99%

Solution Structures of Ca2+-CIB1 and Mg2+-CIB1 and Their Interactions with the Platelet Integrin αIIb Cytoplasmic Domain

Huang

Ishida

Yamniuk

et al. 2011

Journal of Biological Chemistry

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The calcium-and integrin-binding protein 1 (CIB1) is a ubiquitous Ca 2؉ -binding protein and a specific binding partner for the platelet integrin ␣IIb cytoplasmic domain, which confers the key role of CIB1 in hemostasis. CIB1 is also known to be involved in apoptosis, embryogenesis, and the DNA damage response. In this study, the solution structures of both Ca 2؉ -CIB1 and Mg 2؉ -CIB1 were determined using solution-state NMR spectroscopy. The methyl groups of Ile, Leu, and Val were selectively protonated to compensate for the loss of protons due to deuteration. The solution structure of Ca 2؉ -CIB1 possesses smaller opened EF-hands in its C-domain compared with available crystal structures. Ca 2؉ -CIB1 and Mg 2؉ -CIB1 have similar structures, but the N-lobe of Mg 2؉ -CIB1 is slightly more opened than that of Ca 2؉ -CIB1. Additional NMR experiments, such as chemical shift perturbation and methyl group solvent accessibility as measured by a nitroxide surface probe, were carried out to further characterize the structures of Ca 2؉ -CIB1 and Mg 2؉ -CIB1 as well as their interactions with the integrin ␣IIb cytoplasmic domain. NMR measurements of backbone amide proton slow motion (microsecond to millisecond) dynamics confirmed that the C-terminal helix of Ca 2؉ -CIB1 is displaced upon ␣IIb binding. The EF-hand III of both Ca 2؉ -CIB1 and Mg 2؉ -CIB1 was identified to be directly involved in the interaction of CIB1 with ␣IIb. Together, these data illustrate that CIB1 behaves quite differently from related EF-hand regulatory calcium-binding proteins, such as calmodulin or neuronal calcium sensor proteins.The calcium-and integrin-binding protein 1 (CIB1) is a member of the regulatory Ca 2ϩ -binding helix-loop-helix or EFhand protein family (1). CIB1 is a ubiquitous 191-residue (22 kDa) protein with several functions in cell signaling (2, 3). CIB1 has been reported to interact with a number of protein targets, including the platelet integrin ␣IIb subunit (4), sphingosine kinase 1 (5), p21-activated kinase (6), apoptosis signal-regulating kinase (7), polo-like protein kinases (8), and a recently reported new target in the regulation of cardiac hypertrophy, calcineurin B (9). The interaction of CIB1 with the integrin ␣IIb subunit has been studied extensively (10 -13). The integrin ␣IIb subunit usually associates with the integrin ␤3 subunit, and the heterodimeric ␣IIb␤3 protein is involved in both so-called "inside-out" and "outside-in" signaling pathways (3). CIB1 is believed to be capable of specifically binding to the ␣IIb cytoplasmic domain and dissociating the ␣IIb␤3 dimer, which in turn triggers integrin activation (11,12). The interactions between Ca 2ϩ -CIB1 and a fragment of the integrin ␣IIb subunit (residues 983-1008, hereafter referred to as the ␣IIb peptide) have been suggested to mainly involve a hydrophobic pocket in the C-domain of Ca 2ϩ -CIB1 with a dissociation constant in the submicromolar range (10).The calcium-bound CIB1 (Ca 2ϩ -CIB1) structure consists of four helix-loop-helix (EF-hand) Ca 2ϩ -binding m...

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“…The N z exchange experiments (42)(43)(44) were run with an N z mixing period of 230 ms by using a sample that contained detectable amounts of both free and cAMP-bound RI␣ (residues 119-244). However, because the pure cAMP-free state of RI␣ (residues 119-244) is only poorly stable in solution under the experimental conditions used, the cAMP-induced variations in hydrogen exchange rates were measured by perturbing the binding equilibrium just slightly, i.e., by dialyzing out the excess free-cAMP under native conditions so that only minor populations of the cAMP-free state would coexist in dynamic equilibrium with the bound form of the protein (45). The low concentration of the free state and the dynamic exchange of the cAMP ligand between the free and bound proteins ensures the stability of these samples during the measurement of the hydrogen exchange rates (45).…”

Section: Methodsmentioning

confidence: 99%

cAMP activation of PKA defines an ancient signaling mechanism

Das

Esposito

Abu-Abed

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

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113

106

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allostery ͉ NMR ͉ cyclic nucleotide binding domain T he cAMP binding domain (CBD) and cAMP are conserved from bacteria to humans as a ubiquitous signaling mechanism to translate extracellular stress signals into appropriate biological responses (1). The major receptor for cAMP in higher eukaryotes, cAMP-dependent PKA (2), is ubiquitous in mammalian cells where it exists in two forms: the inactive tetrameric holoenzyme and the active dissociated catalytic subunit (Csubunit). In the inactive holoenzyme, two C-subunits are bound to a dimeric regulatory subunit (R-subunit) (Fig. 1a). Upon binding cAMP, the R-subunits undergo a conformational change that unleashes the active C-subunits (3, 4). The Rsubunits are composed of an N-terminal dimerization/docking domain, a flexible linker that includes an autoinhibitory segment, and two tandem CBDs (CBD-A and CBD-B; Fig. 1b) (5). The CBD-A of the isoform I␣ of the R-subunit of PKA (RI␣) contains a noncontiguous ␣-subdomain, which mediates the interactions with the C-subunit and a contiguous ␤-subdomain that forms a ␤-sandwich and contains the cAMP binding pocket (i.e., the phosphate binding cassette or PBC) ( Fig. 1 c and d) (6).Crystal structures of CBD-A of RI␣ in its cAMP-(6) and C-bound (7) states have revealed two very different conformations, highlighting the conformational plasticity of this ancient domain. Although these static crystal structures define two stable end points, questions remain about the allosteric control of the reversible shuttling between the two states. How does the signal generated by cAMP binding to the PBC (Fig. 1 c and d) propagate through a long-range allosteric network that spans both ␣-and ␤-subdomains? Previous analyses (8-16) have led to the proposal of an initial allosteric model in which the ␣-and ␤-subdomains are directly coupled to each other through a salt bridge between E200 and R241 and also possibly through a hydrophobic hinge defined by the L203, I204, and Y229 side-chain cluster (9,11,12). However, mutations (17), sequence conservation analyses (1), structurebased comparisons (1), and genetic screening (18,19) indicate that several other sites, which are not accounted for by the existing model, are also likely to play an active role in the cAMP-mediated activation of PKA. To comprehensively understand this allosteric mechanism, it is therefore necessary to elucidate at high resolution how cAMP remodels the free energy landscape of CBD-A, which serves as the central controlling unit of PKA. For this purpose, we have investigated by NMR RI␣ (residues 119-244), a construct that spans both ␣-and ␤-subdomains of CBD-A and retains high-

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Mapping Allostery through Equilibrium Perturbation NMR Spectroscopy

Cited by 34 publications

References 25 publications

Allosteric Communication between cAMP Binding Sites in the RI Subunit of Protein Kinase A Revealed by NMR

Allosteric Communication between cAMP Binding Sites in the RI Subunit of Protein Kinase A Revealed by NMR

Solution Structures of Ca2+-CIB1 and Mg2+-CIB1 and Their Interactions with the Platelet Integrin αIIb Cytoplasmic Domain

cAMP activation of PKA defines an ancient signaling mechanism

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