EGFR: Tale of the C‐terminal tail

The carboxy terminal tail of epidermal growth factor receptor (EGFR) plays a critical role in the regulation of the enzyme activity of the kinase. There is a good structural model for the mechanism by which the C‐terminal tail proximal to the kinase domain contributes to the negative regulation of the activity. Its conformation in the active state, conversely, has remained elusive due to its dynamic nature. A recently published structure of EGFR kinase domain shows the conformation of the proximal C‐terminal tail in the active kinase. Analysis of this conformational state of the C‐terminal tail is presented, and some of the mutagenesis data is revisited. © 2013 The Protein Society     

Differential requirement of BAK1 C‐terminal tail in development and immunity

BRI_1-ASSOCIATED RECEPTOR KINASE 1(BAK_1) plays critical roles in plant developmental and immune signaling pathways. BAK_1 and a large number of leucine-rich repeat receptor-like kinases(LRR-RLKs)harbor a mysterious carboxyl-terminal tail(CT) beyond their kinase domain. In this study we analyzed the biological significance of this CT region using a unique bak_1 mutant allele which causes deletion of the CT region. We showed that BAK_1 CT promotes its kinase activity and is required for pathogen-associated molecular pattern(PAMP)-triggered immunity, but it is dispensable for brassinosteroid responses and BAK_1/BKK_1-inhibited cell death signaling. Therefore the BAK_1 C-terminal tail is differentially required for its functions in development and immunity.     

Hsp90 is regulated by a switch point in the C‐terminal domain

Heat shock protein 90 (Hsp90) is an abundant, dimeric ATP‐dependent molecular chaperone, and ATPase activity is essential for its in vivo functions. S‐nitrosylation of a residue located in the carboxy‐terminal domain has been shown to affect Hsp90 activity in vivo. To understand how variation of a specific amino acid far away from the amino‐terminal ATP‐binding site regulates Hsp90 functions, we mutated the corresponding residue and analysed yeast and human Hsp90 variants both in vivo and in vitro. Here, we show that this residue is a conserved, strong regulator of Hsp90 functions, including ATP hydrolysis and chaperone activity. Unexpectedly, the variants alter both the C‐terminal and N‐terminal association properties of Hsp90, and shift its conformational equilibrium within the ATPase cycle. Thus, S‐nitrosylation of this residue allows the fast and efficient fine regulation of Hsp90.     

Crystal structure of the C‐terminal domain of mouse TLR9

Toll‐like receptors (TLRs) are important pattern recognition receptors that function in innate immunity. Elucidating the structure and signaling mechanisms of TLR9, a sensor of foreign and endogenous DNA, is essential for understanding its key role in immunity against microbial pathogens as well as in autoimmunity. Abundant evidence suggests that the TLR9‐CTD (C‐terminal domain) by itself is capable of DNA binding and signaling. The crystal structure of unliganded mouse TLR9‐CTD is presented. TLR9‐CTD exhibits one unique feature, a cluster of stacked aromatic and arginine side chains on its concave face. Overall, its structure is most related to the TLR8‐CTD, suggesting a similar mode of ligand binding and signaling. Proteins 2014; 82:2874–2878. © 2014 Wiley Periodicals, Inc.     

Modeling the interaction between the N‐terminal domain of the tumor suppressor p53 and azurin

It is known that the half life of the tumor suppressor p53 can be increased by the interaction with the bacterial protein azurin, resulting in an enhanced anti‐tumoral activity. The understanding of the molecular mechanisms on the basis of this phenomenon can open the way to new anti‐cancer strategies. Some experimental works have given evidence of an interaction between p53 and azurin (AZ); however the binding regions of the proteins are still unknown. Recently, fluorescence studies have shown that p53 partakes in the binding with the bacterial protein by its N‐terminal (NT) domain. Here we have used a computational method to get insight into this interacting mode. The model that we propose for the best complex between AZ and p53 has been obtained from a rigid‐body docking, coupled with a molecular dynamics (MD) simulation, a free energy calculation, and validated by mutagenesis analysis. We have found a high degree of geometric fit between the two proteins that are kept together by several hydrophobic interactions and numerous hydrogen bonds. Interestingly, it has emerged that AZ binds essentially to the helices H_I and H_III of the p53 NT domain, which are also interacting regions for the foremost inhibitor of p53, MDM2. Copyright © 2009 John Wiley & Sons, Ltd.     

Charge neutralization and collapse of the C‐terminal tail of alpha‐synuclein at low pH

Alpha‐synuclein (αS) is the primary component of Lewy bodies, the pathological hallmark of Parkinson's Disease. Aggregation of αS is thought to proceed from a primarily disordered state with nascent secondary structure through intermediate conformations to oligomeric forms and finally to mature amyloid fibrils. Low pH conditions lead to conformational changes associated with increased αS fibril formation. Here we characterize these structural and dynamic changes using solution state NMR measurements of secondary chemical shifts, relaxation parameters, residual dipolar couplings, and paramagnetic relaxation enhancement. We find that the neutralization of negatively charged side‐chains eliminates electrostatic repulsion in the C‐terminal tail of αS and leads to a collapse of this region at low pH. Hydrophobic contacts between the compact C‐terminal tail and the NAC (non‐amyloid‐β component) region are maintained and may lead to the formation of a globular domain. Transient long‐range contacts between the C‐terminus of the protein and regions N‐terminal to the NAC region are also preserved. Thus, the release of long‐range contacts does not play a role in the increased aggregation of αS at low pH, which we instead attribute to the increased hydrophobicity of the protein.     

Structural and functional analysis of the phosphoryl transfer reaction mediated by the human small C‐terminal domain phosphatase,Scp1

Human small C‐terminal domain phosphatase 1 (Scp1) modulates the phosphorylation state of the C‐terminal domain (CTD) of eukaryotic RNA polymerase II (RNAP II), with preference for phosphorylated Ser5 in the tandem heptad repeats of the CTD. Additionally, Scp1 was identified as a conserved regulator of neuronal stem cell development. Scp1 is a member of haloacid dehalogenase (HAD) superfamily, whose catalysis depends on a Mg²⁺ ion and a DXDX(T/V) motif. The first Asp of the motif is identified as the nucleophile that is subject to phosphorylation leading to a phosphoryl‐aspartate intermediate. This high‐energy mixed anhydride intermediate is subsequently hydrolyzed to regenerate the enzyme. In the present study, we successfully captured the phosphoryl‐aspartate intermediate in the crystal structure of a Scp1D206A mutant soaked with para‐nitrophenyl phosphate (pNPP), providing strong evidence for the proposed mechanism. Furthermore, steady‐state kinetic analysis of a variety of Scp1 mutants revealed the importance of Asp206 in Mg²⁺ coordination mediated by a water molecule. Overall, we captured the snapshots of the phosphoryl transfer reaction at each stage of Scp1‐mediated catalysis. Through structural‐based sequence alignment, we show that the spatial position of the D206 side chain is strictly conserved throughout HAD family. Our results strongly suggest that Asp206 and its equivalent residues in other HAD family members play important structural and possible mechanistic roles.     

Solution structure of the calmodulin‐like C‐terminal domain of Entamoeba α‐actinin2

Cell motility is dependent on a dynamic meshwork of actin filaments that is remodelled continuously. A large number of associated proteins that are severs, cross‐links, or caps the filament ends have been identified and the actin cross‐linker α‐actinin has been implied in several important cellular processes. In Entamoeba histolytica, the etiological agent of human amoebiasis, α‐actinin is believed to be required for infection. To better understand the role of α‐actinin in the infectious process we have determined the solution structure of the C‐terminal calmodulin‐like domain using NMR. The final structure ensemble of the apo form shows two lobes, that both resemble other pairs of calcium‐binding EF‐hand motifs, connected with a mobile linker. Proteins 2016; 84:461–466. © 2016 Wiley Periodicals, Inc.     

Structural basis for regulation of poly‐SUMO chain by a SUMO‐like domain of Nip45

Post‐translational modification by small ubiquitin‐like modifier (SUMO) provides an important regulatory mechanism in diverse cellular processes. Modification of SUMO has been shown to target proteins involved in systems ranging from DNA repair pathways to the ubiquitin‐proteasome degradation system by the action of SUMO‐targeted ubiquitin ligases (STUbLs). STUbLs recognize target proteins modified with a poly‐SUMO chain through their SUMO‐interacting motifs (SIMs). STUbLs are also associated with RENi family proteins, which commonly have two SUMO‐like domains (SLD1 and SLD2) at their C terminus. We have determined the crystal structures of SLD2 of mouse RENi protein, Nip45, in a free form and in complex with a mouse E2 sumoylation enzyme, Ubc9. While Nip45 SLD2 shares a β‐grasp fold with SUMO, the SIM interaction surface conserved in SUMO paralogues does not exist in SLD2. Biochemical data indicates that neither tandem SLDs or SLD2 of Nip45 bind to either tandem SIMs from either mouse STUbL, RNF4 or to those from SUMO‐binding proteins, whose interactions with SUMO have been well characterized. On the other hand, Nip45 SLD2 binds to Ubc9 in an almost identical manner to that of SUMO and thereby inhibits elongation of poly‐SUMO chains. This finding highlights a possible role of the RENi proteins in the modulation of Ubc9‐mediated poly‐SUMO formation. Proteins 2010. © 2009 Wiley‐Liss, Inc.     

The structure of S. lividans acetoacetyl‐CoA synthetase shows a novel interaction between the C‐terminal extension and the N‐terminal domain

The adenosine monoposphate‐forming acyl‐CoA synthetase enzymes catalyze a two‐step reaction that involves the initial formation of an acyl adenylate that reacts in a second partial reaction to form a thioester between the acyl substrate and CoA. These enzymes utilize a Domain Alternation catalytic mechanism, whereby a ～110 residue C‐terminal domain rotates by 140° to form distinct catalytic conformations for the two partial reactions. The structure of an acetoacetyl‐CoA synthetase (AacS) is presented that illustrates a novel aspect of this C‐terminal domain. Specifically, several acetyl‐ and acetoacetyl‐CoA synthetases contain a 30‐residue extension on the C‐terminus compared to other members of this family. Whereas residues from this extension are disordered in prior structures, the AacS structure shows that residues from this extension may interact with key catalytic residues from the N‐terminal domain. Proteins 2015; 83:575–581. © 2014 Wiley Periodicals, Inc.     

C‐terminal domain of SARS‐CoV main protease can form a 3D domain‐swapped dimer

SARS coronavirus main protease (M^pro) plays an essential role in the extensive proteolytic processing of the viral polyproteins (pp1a and pp1ab), and it is an important target for anti-SARS drug development. We have reported that both the M^pro C-terminal domain alone (M^pro-C) and the N-finger deletion mutant of M^pro (M^pro-Δ7) exist as a stable dimer and a stable monomer (Zhong et al., J Virol 2008; 82:4227-4234). Here, we report structures of both M^pro-C monomer and dimer. The structure of the M^pro-C monomer is almost identical to that of the C-terminal domain in the crystal structure of M^pro. Interestingly, the M^pro-C dimer structure is characterized by 3D domain-swapping, in which the first helices of the two protomers are interchanged and each is enwrapped by four other helices from the other protomer. Each folding subunit of the M^pro-C domain-swapped dimer still has the same general fold as that of the M^pro-C monomer. This special dimerization elucidates the structural basis for the observation that there is no exchange between monomeric and dimeric forms of M^pro-C and M^pro-Δ7.     

Structural insight into the interaction of proteins containing NPF,DPF, and GPF motifs with the C‐terminal EH‐domain of EHD1

Eps15 homology (EH)‐domain containing proteins are regulators of endocytic membrane trafficking. EH‐domain binding to proteins containing the tripeptide NPF has been well characterized, but recent studies have shown that EH‐domains are also able to interact with ligands containing DPF or GPF motifs. We demonstrate that the three motifs interact in a similar way with the EH‐domain of EHD1, with the NPF motif having the highest affinity due to the presence of an intermolecular hydrogen bond. The weaker affinity for the DPF and GPF motifs suggests that if complex formation occurs in vivo, they may require high ligand concentrations, the presence of successive motifs and/or specific flanking residues.     

C‐terminal tail derived from the neighboring subunit is critical for the activity of Thermoplasma acidophilumD‐aldohexose dehydrogenase

The D ‐aldohexose dehydrogenase from the thermoacidophilic archaeon Thermoplasma acidophilum (AldT) is a homotetrameric enzyme that catalyzes the oxidation of several D ‐aldohexoses, especially D ‐mannose. AldT comprises a unique C‐terminal tail motif (residues 247–255) that shuts the active‐site pocket of the neighboring subunit. The functional role of the C‐terminal tail of AldT has been investigated using mutational and crystallographic analyses. A total of four C‐terminal deletion mutants (Δ254, Δ253, Δ252, and Δ249) and two site‐specific mutants (Y86G and P254G) were expressed by Escherichia coli and purified. Enzymatic characterization of these mutants revealed that the C‐terminal tail is a requisite and that the interaction between Tyr86 and Pro254 is critical for enzyme activity. The crystal structure of the Δ249 mutant was also determined. The structure showed that the active‐site loops undergo a significant conformational change, which leads to the structural deformation of the substrate‐binding pocket. Proteins 2009. © 2008 Wiley‐Liss, Inc.     

Functional cross-talk of HIV-1 Tat with p53 through its C-terminal domain

Human immunodeficiency virus type 1 (HIV-1) Tat repressed the p53-dependent gene expression through its C-terminal domain of Tat (amino acid residues 73-86) independent of the involvement of NF-kappaB and coactivator CBP/p300. Although Tat did not directly bind to p53, this repression required the N-terminal domain of p53. In contrast, Tat and p53 cooperated in the activation of HIV-1 gene expression. Thus, the cross-talk between Tat and p53 may be linked with cellular transformation by HIV-1 infection or activation of HIV-1 replication.     

Structure and interactions of the C‐terminal metal binding domain of Archaeoglobus fulgidus CopA

The Cu⁺‐ATPase CopA from Archaeoglobus fulgidus belongs to the P_1B family of the P‐type ATPases. These integral membrane proteins couple the energy of ATP hydrolysis to heavy metal ion translocation across membranes. A defining feature of P_1B‐1‐type ATPases is the presence of soluble metal binding domains at the N‐terminus (N‐MBDs). The N‐MBDs exhibit a conserved ferredoxin‐like fold, similar to that of soluble copper chaperones, and bind metal ions via a conserved CXXC motif. The N‐MBDs enable Cu⁺ regulation of turnover rates apparently through Cu‐sensitive interactions with catalytic domains. A. fulgidus CopA is unusual in that it contains both an N‐terminal MBD and a C‐terminal MBD (C‐MBD). The functional role of the unique C‐MBD has not been established. Here, we report the crystal structure of the apo, oxidized C‐MBD to 2.0 Å resolution. In the structure, two C‐MBD monomers form a domain‐swapped dimer, which has not been observed previously for similar domains. In addition, the interaction of the C‐MBD with the other cytoplasmic domains of CopA, the ATP binding domain (ATPBD) and actuator domain (A‐domain), has been investigated. Interestingly, the C‐MBD interacts specifically with both of these domains, independent of the presence of Cu⁺ or nucleotides. These data reinforce the uniqueness of the C‐MBD and suggest a distinct structural role for the C‐MBD in CopA transport. Proteins 2010. © 2010 Wiley‐Liss, Inc.