An Insight into the Biological Functions, the Molecular Mechanism and the Nature of Interactions of a Set of Biologically Important Proteins.

Characterization of protein structural properties is crucial to determine its role in normal and pathological conditions. In this dissertation, we have employed NMR spectroscopy in a combination of other biochemical and biophysical techniques to investigate the catalytic function, the molecular mechanism, and nature of the interactions of bmAANAT3, Cdc37, and TIMP2, respectively. In the first project, we have employed an arylalkylamine N-acetyltransferases (AANATs) from Bombyx mori (bmAANAT3) to determine the role of the acetyl-group in coordinating the catalytic cycle in this family of enzymes. We have found that the occupancy of the acetyl-moiety in the catalytic funnel of these enzymes coordinates the ordered progress of their catalytic cycle by regulating the conformational properties of their acetyl-group donor and acceptor binding sites. In the second project, the catalytic domain of the Hsp90 client kinase bRaf has been used to investigate the role of kinome specific Hsp90 cochaperone Cdc37 in the Hsp90 chaperone machinery. Our data has revealed that Cdc37 sorts a broad range of kinases into Hsp90 clients and nonclients by scanning their thermal stability through locally unfolding only the client kinases. Finally, we have used the N-terminal domain of the tissue inhibitor metalloproteinase-2 (N-TIMP2) to provide an insight into the extracellular interaction activity of Hsp90. Our data has shown that both Hsp90 and its middle domains (mHsp90) interact with N-TIMP2 in a transient low-affinity regime. However, in both states of the Hsp90, this interaction is driven by a subset of hydrophobic residues located at the surface of N-TIMP2

Acetyl group coordinated progression through the catalytic cycle of an arylalkylamine N-acetyltransferase

<div><p>The transfer of an acetyl group from acetyl-CoA to an acceptor amine is a ubiquitous biochemical transformation catalyzed by Gcn5-related N-acetyltransferases (GNATs). Although it is established that the reaction proceeds through a sequential ordered mechanism, the role of the acetyl group in driving the ordered formation of binary and ternary complexes remains elusive. Herein, we show that CoA and acetyl-CoA alter the conformation of the substrate binding site of an arylalkylamine N-acetyltransferase (AANAT) to facilitate interaction with acceptor substrates. However, it is the presence of the acetyl group within the catalytic funnel that triggers high affinity binding. Acetyl group occupancy is relayed through a conserved salt bridge between the P-loop and the acceptor binding site, and is manifested as differential dynamics in the CoA and acetyl-CoA-bound states. The capacity of the acetyl group carried by an acceptor to promote its tight binding even in the absence of CoA, but also its mutually exclusive position to the acetyl group of acetyl-CoA underscore its importance in coordinating the progression of the catalytic cycle.</p></div

Molecular Mechanism of Protein Kinase Recognition and Sorting by the Hsp90 Kinome-Specific Cochaperone Cdc37

Despite the essential functions of Hsp90, little is known about the mechanism that controls substrate entry into its chaperone cycle. We show that the role of Cdc37 cochaperone reaches beyond that of an adaptor protein and find that it participates in the selective recruitment of only client kinases. Cdc37 recognizes kinase specificity determinants in both clients and nonclients and acts as a general kinase scanning factor. Kinase sorting within the client-to-nonclient continuum relies on the ability of Cdc37 to challenge the conformational stability of clients by locally unfolding them. This metastable conformational state has high affinity for Cdc37 and forms stable complexes through a multidomain cochaperone interface. The interaction with nonclients is not accompanied by conformational changes of the substrate and results in substrate dissociation. Collectively, Cdc37 performs a quality control of protein kinases, where induced conformational instability acts as a flag for Hsp90 dependence and stable cochaperone association

Molecular Mechanism of Protein Kinase Recognition and Sorting by the Hsp90 Kinome-Specific Cochaperone Cdc37

Despite the essential functions of Hsp90, little is known about the mechanism that controls substrate entry into its chaperone cycle. We show that the role of Cdc37 cochaperone reaches beyond that of an adaptor protein and find that it participates in the selective recruitment of only client kinases. Cdc37 recognizes kinase specificity determinants in both clients and nonclients and acts as a general kinase scanning factor. Kinase sorting within the client-to-nonclient continuum relies on the ability of Cdc37 to challenge the conformational stability of clients by locally unfolding them. This metastable conformational state has high affinity for Cdc37 and forms stable complexes through a multidomain cochaperone interface. The interaction with nonclients is not accompanied by conformational changes of the substrate and results in substrate dissociation. Collectively, Cdc37 performs a quality control of protein kinases, where induced conformational instability acts as a flag for Hsp90 dependence and stable cochaperone association

Cofactor-mediated conformational remodeling of <i>bm</i>AANAT3 monitored by NMR.

<p>(A) acetyl-CoA induced CSPs mapped on the model of <i>bm</i>AANAT3. (B) CSPs as a function of primary sequence for the interaction of <i>bm</i>AANAT3 with acetyl-CoA (green) and CoA (cyan). CSPs greater than the mean or one SD above the mean are marked by continuous and broken lines, respectively. The conserved motifs of the GNAT superfamily are indicated on the left. Black bars in the acetyl-CoA portion of the plot mark G135 and G137 which are absent in the free state of the enzyme, but tentatively assigned in the bound state. Red bars in the CoA portion of the plot mark residues that become broadened beyond detection in the CoA-bound state. (C) CoA induced CSPs mapped on the model of <i>bm</i>AANAT3. In (A) and (C) the white region in the three-color gradient corresponds to the mean CSP, while the magenta region to greater than mean + σ. Residues highlighted in orange in (C) highlight residues that broaden beyond detection in the presence of CoA. The position of acetyl-CoA is highlighted in green, after aligning the <i>bm</i>AANAT3 model with the structure of acetyl-CoA-bound DAT. Residues that experience significant CSPs for acetyl-CoA and CoA are listed in (A), while, signals that broaden (beyond detection) upon CoA binding are listed in (C).</p

Association of <i>bm</i>AANAT3 with acetyl-CoA and CoA followed by NMR.

<p>(A) ¹⁵N-HSQC of <i>bm</i>AANAT3 in the free (blue) and acetyl-CoA-bound state (green). The very large number of signals affected by acetyl-CoA indicates that chemical shift perturbations do not solely report cofactor binding at the catalytic funnel, but also induced conformational changes to distal regions of the enzyme. (B) Expanded region of the ¹⁵N-HSQC of <i>bm</i>AANAT3 in the free-state (blue), after the addition of 0.5 (orange) or 1.0 (green) equivalents of acetyl-CoA, showing the concomitant disappearance and appearance of the signals for the free and bound states. (C) Expanded region from the NMR titration course of <i>bm</i>AANAT3 with CoA highlighting signals from residues that either do not exhibit any line broadening (L76) or broaden beyond detection at different points of the titration (R125, D161, A162 and L179).</p

An overview of dissociation constants measured in this study.

<p>For those complexes that gave poor <i>c-values</i> in the ITC, K_d values were determined by NMR (marked by *). A summary of K_d values is presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0177270#pone.0177270.t001" target="_blank">Table 1</a>. (A) Determination of K_d values for the interaction of free <i>bm</i>AANAT3 with acetyl-CoA (black) and CoA (gray), and DAT with acetyl-CoA (red). The isotherms presented in the middle panel correspond to interaction of <i>bm</i>AANAT3 with acetyl-CoA and CoA (black) and to the interaction of DAT with acetyl-CoA (red). NMR was used to confirm the K_d of <i>bm</i>AANAT3 for CoA (right panel). (B) Determination of K_d values for the interaction of different cofactor-liganded states of <i>bm</i>AANAT3 with tryptamine and tryptamine analogs. The presence of acetyl-CoA in the catalytic funnel leads to an increase in the affinity for tryptophol, while an even more substantial increase is observed when an acetylated derivative of tryptamine (N-acetyltryptamine) is utilized. Binding of N-acetyltryptamine to <i>bm</i>AANAT3 is not sensitive to the presence of CoA, but it is abolished in the presence of acetyl-CoA. The isotherms presented in the middle panel correspond to the interaction of <i>bm</i>AANAT3 with N-acetyltryptamine in the free- or CoA-bound states (black) and in the acetyl-CoA-bound state (red). NMR was used to measure the K_d for the interaction of free- and acetyl-CoA-bound <i>bm</i>AANAT3 with tryptophol (right panel). (C) Determination of K_d values for the interaction of different cofactor-liganded states of <i>bm</i>AANAT3 and variants (D26A) with tyramine and tyramine analogs. The presence of CoA in the catalytic funnel leads to an increase in the affinity for tyrosol, while an even more substantial increase is observed in the presence of acetyl-CoA. The D26A mutation renders tyrosol binding insensitive to acetyl-group occupancy of the catalytic center, in a manner that tyrosol affinity of acetyl-CoA-bound <i>bm</i>AANAT3^D26A is similar to that of CoA-bound <i>bm</i>AANAT3^D26A or CoA-bound <i>bm</i>AANAT3. NMR was used to measure the K_d for the interaction of tyrosol with free- or CoA-bound <i>bm</i>AANAT3, and for acetyl-CoA- or CoA-bound <i>bm</i>AANAT3^D26A.</p

Assembly of <i>bm</i>AANAT3 complexes with acceptor substrates monitored by NMR.

<p>(A) Mapping of tryptophol induced CSPs to free <i>bm</i>AANAT3. The conserved motifs of the GNAT superfamily are indicated on the left. (B) CSPs as a function of primary sequence for the interaction of tryptophol with free- (blue) and acetyl-CoA-bound <i>bm</i>AANAT3 (red). CSPs greater than the mean or one SD above the mean are marked by continuous and broken lines, respectively. Green, open bars in the free-state mark residues that disappear in the substrate-bound state. (C) Mapping of tryptophol induced CSPs to acetyl-CoA bound <i>bm</i>AANAT3. The bisubstrate, CoA-S-acetyltryptamine (green), was modelled by aligning <i>bm</i>AANAT3 model to the structure of <i>oa</i>AANAT.</p

Dissociation constants measured for <i>bm</i>AANAT3, in μM.

<p>Dissociation constants measured for <i>bm</i>AANAT3, in μM.</p