Protein Interaction Domains Research Articles

Identification of a second binding site on the TRIM25 B30.2 domain.

The retinoic acid-inducible gene-I (RIG-I) receptor recognizes short 5'-di- and triphosphate base-paired viral RNA and is a critical mediator of the innate immune response against viruses such as influenza A, Ebola, HIV and hepatitis C. This response is reported to require an orchestrated interaction with the tripartite motif 25 (TRIM25) B30.2 protein-interaction domain. Here, we present a novel second RIG-I-binding interface on the TRIM25 B30.2 domain that interacts with CARD1 and CARD2 (caspase activation and recruitment domains) of RIG-I and is revealed by the removal of an N-terminal α-helix that mimics dimerization of the full-length protein. Further characterization of the TRIM25 coiled-coil and B30.2 regions indicated that the B30.2 domains move freely on a flexible tether, facilitating RIG-I CARD recruitment. The identification of a dual binding mode for the TRIM25 B30.2 domain is a first for the SPRY/B30.2 domain family and may be a feature of other SPRY/B30.2 family members.

Abstract IA20: Autophagy in alveolar soft part sarcomagenesis

Abstract Altered metabolism is considered to be one of the new hallmarks of cancer. Autophagy is one major avenue of altered cancer metabolism, enabling cell survival under metabolic stress and promoting chemoresistance. The nuclear localization of MiTF/TFE3 family transcription factors has associated with upregulated transcription of autophagy genes in pancreatic cancer. Alveolar soft part sarcoma is a rare but deadly soft-tissue sarcoma, with a predilection for adolescent and young adult victims. Alveolar soft part sarcoma is noteworthy for its resistance to traditional cytotoxic chemotherapies. It consistently associates with a t(X;17) chromosomal translocation that produces the ASPSCR1-TFE3 target gene, bearing the DNA-binding domain from TFE3 and protein interaction domains from ASPSCR1. We have demonstrated that conditional expression of ASPSCR1-TFE3 is sufficient to drive alveolar soft part sarcomagenesis in the mouse with complete penetrance. Mouse tumors recapitulate human alveolar soft part sarcoma histology and transcriptomes faithfully. While direct targets of ASPSCR1-TFE3 have been studied in a renal cell carcinoma cell line, they have not been studied in alveolar soft part sarcoma. We report not only the first genome-wide localization of the ASPSCR1-TFE3 oncoprotein on chromatin from alveolar soft part sarcoma cell lines and mouse tumors, but also its association with actively transcribed genes. Among these are found many genes related to autophagy, especially those related specifically to the nutrient responsive pathways that drive autophagy. We demonstrate high expression of autophagy-related lysosomes and proteins at baseline conditions in human tumors and cell lines and mouse tumors. We also demonstrate active autophagic flux even in the absence of stress conditions. Inhibition of autophagy has no apparent impact on the survival of alveolar soft part sarcoma cells alone, but profoundly impacts their protein degradation pathways and the availability of amino acids for protein assembly in stress. Inhibition of autophagy strongly synergizes with chemotherapy to kill alveolar soft part sarcoma cells, suggesting it was a source mechanism for resistance. We have therefore demonstrated the direct targets of ASPSCR1-TFE3 in alveolar soft part sarcomas, including a number of autophagy genes that are expressed in these tumors, independently from nutrient deprivation or stress, rendering cells particularly resistant to many therapy-induced stresses. Citation Format: Kevin B. Jones. Autophagy in alveolar soft part sarcomagenesis [abstract]. In: Proceedings of the AACR Conference on Advances in Sarcomas: From Basic Science to Clinical Translation; May 16-19, 2017; Philadelphia, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(2_Suppl):Abstract nr IA20.

Analyzing Protein Domain Interactions in Chemoreceptors by In Vivo PEGylation.

The instability of some proteins can hamper in vitro studies. This is true for the membrane-bound aerotaxis receptor, Aer, which exhibits significant proteolysis during the preparation of membrane vesicles. Permeabilized cells can closely mimic in vivo conditions, maintaining the intracellular milieu and geometry of interacting domains. Here, we describe an optimized method for determining solvent accessibility in permeabilized Escherichia coli cells. In this method, E. coli expressing Aer with a series of cysteine replacements are treated with toluene and ethanol, after which a large sulfhydryl reactive probe, PEG-mal, is added. PEGylated protein is separated from un-PEGylated protein by its apparent size difference on SDS-PAGE. The extent to which each cysteine residue becomes PEGylated is then used as a measure of solvent accessibility. When a library of single-Cys replacements is mapped, regions of low accessibility can suggest interacting protein surfaces. We successfully used this method to reveal inaccessible surfaces on both the Aer PAS and HAMP domains that were then shown by disulfide cross-linking to interact.

Structure function relations in PDZ-domain-containing proteins: Implications for protein networks in cellular signalling

Protein scaffolds as essential backbones for organization of supramolecular signalling complexes are a recurrent theme in several model systems. Scaffold proteins preferentially employ linear peptide binding motifs for recruiting their interaction partners. PDZ domains are one of the more commonly encountered peptide binding domains in several proteins including those involved in scaffolding functions. This domain is known for its promiscuity both in terms of ligand selection, mode of interaction with its ligands as well as its association with other protein interaction domains. PDZ domains are subject to several means of regulations by virtue of their functional diversity. Additionally, the PDZ domains are refractive to the effect of mutations and maintain their three-dimensional architecture under extreme mutational load. The biochemical and biophysical basis for this selectivity as well as promiscuity has been investigated and reviewed extensively. The present review focuses on the plasticity inherent in PDZ domains and its implications for modular organization as well as evolution of cellular signalling pathways in higher eukaryotes.

Ankyrin repeats as a dimerization module

Legionella pneumophila is a pathogen, causing severe pneumonia in humans called Legionnaires' disease. AnkC (LegA12) is a poorly characterized 495-residue effector protein conserved in multiple Legionella species. Here, we report the crystal structure of a C-terminally truncated AnkC (2–384) at 3.2 Å resolution. The structure shows seven ankyrin repeats (ARs) with unique structural features. AnkC forms a dimer along the outer surface of loops between ARs. The dimer exists both in the crystal form and in solution, as shown by analytical ultracentrifugation. This is the first example of ARs as a dimerization module as opposed to solely a protein interaction domain. In addition, a novel α-helix insert between AR3-AR4 is positioned across the surface opposite the ankyrin groove. Sequence conservation suggests that the ankyrin groove of AnkC is a functional site that interacts with binding targets. This ankyrin domain structure is an important step towards a functional characterization of AnkC.

Identification of the interaction and interaction domains of chicken anemia virus VP2 and VP3 proteins

Chicken anemia virus (CAV) is a small, single-stranded DNA virus of Anelloviridae family. Its genome segments encode three proteins, VP1, VP2, and VP3. This study identified an interaction between VP2 and VP3 and mapped the interaction domains. Through the yeast two-hybrid (Y2H) system, VP2 was found to interact with VP3. The presence of the VP2–VP3 complex in CAV-infected chicken cells was confirmed by co-immunoprecipitation. Confocal microscopy showed that VP2 and VP3 were expressed in the cytoplasm in cotransfected Vero cells. In the Y2H system, the interaction domains were identified as being within the N-terminal aa 1–30 and C-terminal aa 17–60 for VP2 and the N-terminal aa 46–60 and C-terminal aa 1–7 for VP3. This study showed the interaction between VP2 and VP3 of CAV and identified multiple independent interactive domains within the two proteins. This provides novel information for investigating the biological functions of these proteins.

Facilitating identification of minimal protein binding domains by cross-linking mass spectrometry

Characterization of protein interaction domains is crucial for understanding protein functions. Here we combine cross-linking mass spectrometry (XL-MS) with deletion analysis to accurately locate minimal protein interaction domains. As a proof of concept, we investigated in detail the binding interfaces of two protein assemblies: the complex formed by MICAL3, ELKS and Rab8A, which is involved in exocytosis, and the complex of SLAIN2, CLASP2 and ch-TOG, which controls microtubule dynamics. We found that XL-MS provides valuable information to efficiently guide the design of protein fragments that are essential for protein interaction. However, we also observed a number of cross-links between polypeptide regions that were dispensable for complex formation, especially among intrinsically disordered sequences. Collectively, our results indicate that XL-MS, which renders distance restrains of linked residue pairs, accelerates the characterization of protein binding regions in combination with other biochemical approaches.

Abstract PR16: A druggable transcriptional vulnerability in NRF2-dependent lung cancer

Abstract The b-ZIP transcription factor NRF2 is a master regulator of the cellular antioxidant response and frequently activated in Non-Small Cell Lung Cancers (NSCLCs). While direct pharmacological inhibition of NRF2 has proven challenging, its aberrant activation rewires metabolic and signaling networks in cancer cells that may create special vulnerabilities for therapeutic intervention. Here, we use chemical proteomics to map ligandable proteins in NRF2-activated NSCLC cells, leading to the discovery of metabolic and transcriptional pathways that are strictly regulated by NRF2. Principal among these was the orphan nuclear receptor NR0B1, which we show nucleates a multimeric protein complex required for the transcriptional output and anchorage-independent growth of NRF2-driven cancers. We further identify small-molecules that target a conserved cysteine within NR0B1's protein interaction domain and show that these chemical probes disrupt NR0B1 complexes and NRF2-dependent cancer cell growth. Our findings thus designate NR0B1 as a druggable, transcriptional co-dependency target for NRF2-activated lung cancers. Citation Format: Liron Bar-Peled, Esther Kemper, Benjamin Cravatt. A druggable transcriptional vulnerability in NRF2-dependent lung cancer [abstract]. In: Proceedings of the AACR Precision Medicine Series: Opportunities and Challenges of Exploiting Synthetic Lethality in Cancer; Jan 4-7, 2017; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2017;16(10 Suppl):Abstract nr PR16.

Chemical Proteomics Identifies Druggable Vulnerabilities in a Genetically Defined Cancer

The transcription factor NRF2 is a master regulator of the cellular antioxidant response, and it is often genetically activated in non-small-cell lung cancers (NSCLCs) by, for instance, mutations in the negative regulator KEAP1. While direct pharmacological inhibition of NRF2 has proven challenging, its aberrant activation rewires biochemical networks in cancer cells that may create special vulnerabilities. Here, we use chemical proteomics to map druggable proteins that are selectively expressed in KEAP1-mutant NSCLC cells. Principal among these is NR0B1, an atypical orphan nuclear receptor that we show engages in a multimeric protein complex to regulate the transcriptional output of KEAP1-mutant NSCLC cells. We further identify small molecules that covalently target a conserved cysteine within the NR0B1 protein interaction domain, and we demonstrate that these compounds disrupt NR0B1 complexes and impair the anchorage-independent growth of KEAP1-mutant cancer cells. Our findings designate NR0B1 as a druggable transcriptional regulator that supports NRF2-dependent lung cancers.

TH.I.9 - Development of microdystrophins for gene therapy of DMD

Gene therapy represents a promising approach to treat DMD as it has the potential to circumvent the primary cause of the disorder, a mutant gene. Gene replacement therapies have focused on methods to deliver expression cassettes encoding the dystrophin protein. Challenges to this approach have included the enormous size of the dystrophin gene (2.2 MB), its muscle mRNA (14 kb) and difficulties in safely delivering vectors to muscles bodywide. We and others have shown that vectors derived from specific serotypes of adeno-associated virus (AAV) can systemically deliver genes to muscles after intravascular infusion. However, AAV vectors have a limited carrying capacity of approximately 5 kb, necessitating the design of miniaturized cDNAs and gene regulatory elements that enable expression of “microdystrophins”. Early generation microdystrophins displayed functional limitations due to their small size (<4 kb) and the absence of some protein interaction domains. To develop improved vectors we tested a variety of novel microdystrophins lacking the C-terminal domain but carrying 4–6 spectrin-like repeats, including the nitric oxide synthase localization domain. We also tested a set of powerful gene regulatory elements based on the muscle creatine kinase (MCK) enhancer plus promoter. These novel microdystrophins were evaluated by AAV-mediated delivery to dystrophic mdx4cv mice followed by extensive analysis of muscle pathophysiology. We identified a 5 repeat vector that when driven by the CK8 regulatory cassette leads to stable expression and profound reductions in muscle pathophysiology. This AAV/CK8-µDys5 vector performs better than previously tested microdystrophin cassettes and represents a promising clinical candidate. We have also been testing AAV vectors to deliver components of the CRISPR/Cas9 system as a method to potentially repair, rather than replace, the mutant dystrophin gene. While this gene editing approach shows promise, its efficiency remains low and as currently applied is less effective than direct delivery of microd

Selective Targeting of Bromodomains of the Bromodomain-PHD Fingers Family Impairs Osteoclast Differentiation.

Histone acetyltransferases of the MYST family are recruited to chromatin by BRPF scaffolding proteins. We explored functional consequences and the therapeutic potential of inhibitors targeting acetyl-lysine dependent protein interaction domains (bromodomains) present in BRPF1–3 in bone maintenance. We report three potent and selective inhibitors: one (PFI-4) with high selectivity for the BRPF1B isoform and two pan-BRPF bromodomain inhibitors (OF-1, NI-57). The developed inhibitors displaced BRPF bromodomains from chromatin and did not inhibit cell growth and proliferation. Intriguingly, the inhibitors impaired RANKL-induced differentiation of primary murine bone marrow cells and human primary monocytes into bone resorbing osteoclasts by specifically repressing transcriptional programs required for osteoclastogenesis. The data suggest a key role of BRPF in regulating gene expression during osteoclastogenesis, and the excellent druggability of these bromodomains may lead to new treatment strategies for patients suffering from bone loss or osteolytic malignant bone lesions.

Removal of a consensus proline is not sufficient to allow tetratricopeptide repeat oligomerization.

Tetratricopeptide repeat (TPR) domains are ubiquitous protein interaction domains that adopt a modular antiparallel array of α-helices. The TPR fold typically adopts a monomeric state, and consensus TPRs sequences successfully fold into the expected monomeric topology. The versatility of the TPR fold also supports different quaternary structures, which may function as regulatory switches. One example is yeast mitochondrial fission 1 (Fis1) that appears to interconvert between monomer and dimer states in regulating division of peroxisomes and mitochondria. Whether human Fis1 can also interconvert like the yeast molecule is unknown. A TPR consensus proline residue present in human Fis1 is absent in the yeast molecule and, when added, prevents yeast Fis1 dimerization suggesting that the TPR consensus proline might have persisted to prevent TPR oligomerization. Here, we address this question with human Fis1 and the consensus TPR protein CTPR3. We demonstrate that human Fis1 does not form a noncovalent dimer via its TPR domain, despite conditions that favor dimerization of the yeast protein. We also show that the presence of the consensus proline is not sufficient to forbid TPR dimerization. Lastly, an analysis of all available TPR protein structures (22 nonredundant structures, totaling 64 TPRs-42 with the consensus proline and 22 without) revealed that the consensus proline is not necessary for turn formation, but does favor shorter turns. This work suggests the TPR consensus proline is not to prevent oligomerization, but to favor tight turns between repeats.

Abstract 449: The H2 Domain in the Connexin43 Carboxyl Terminus Regulates Cardioprotection From Ischemia Reperfusion Injury

Cardiac ischemia-reperfusion (IR) injury occurs in patients with acute coronary syndromes undergoing percutaneous intervention to re-establish blood flow in blocked or narrowed arteries. Presently, there are no therapies approved to prevent the loss of heart muscle, scar tissue formation and contractile dysfunction that result from IR injury. We have previously reported that α CT1, a Connexin 43 (Cx43) mimetic peptide currently in Phase III testing in humans (GAIT1; NCT02667327) as a therapy for chronic skin wounds (PMID:27856288; 25072595), improves contractile function of mouse hearts following injury in association with increases in PKCε-mediated phosphorylation of Cx43 at serine 368 (S368) (PMID: 21273554). Here, we report that induction of S368 phosphorylation and cardioprotection from IR injury by α CT1 involves direct protein-protein interaction of α CT1 with a short alpha-helical sequence within the Cx43 carboxyl terminus (CT) termed the H2 domain. The interacting protein domains involved in Cx43 S368 phosphorylation were identified and validated by a combination of techniques including tandem mass spectroscopy, surface plasmon resonance, immunoprecipitation and thermal shift assays. These studies indicated that α CT1 induced S368 phosphorylation (pS368) via a novel mechanism involving interaction between a pair of positively charged lysines (K344, K345) in the Cx43 CT H2 domain and a cluster of negatively charged amino acids i α CT1. It is further shown that the known ability of α CT1 to interact with Zonula Occludens-1 (ZO-1), has no direct role in either the induction of S368 phosphorylation or protection from IR injury. Finally, we identify a small, novel variant of α CT1 ( α CT11 - RPRPDDLEI, Mr 1110) that penetrates myocyte membranes, induces S368 phosphorylation and robustly preserves ventricular contractile function and cardiac muscle when administered after ischemic injury. Data on Cx43 hemichannel dependence of α CT11 uptake by cells will be presented. Our results indicate that selective targeting of the secondary structure and phospho-status of the Cx43 CT by small peptidergic drugs may provide a novel and potent pharmacological approach to preservation of cardiac muscle following IR injury.

Remarkable Evolutionary Conservation of Antiobesity ADIPOSE/WDTC1 Homologs in Animals and Plants.

ASG2 (Altered Seed Germination 2) is a prenylated protein in Arabidopsis thaliana that participates to abscisic acid signaling and is proposed to act as a substrate adaptor for the DDB1 (DNA damage-binding protein 1)-CUL4 (Cullin 4) E3 ubiquitin ligase complex. ASG2 harbors WD40 and TetratricoPeptide Repeat (TPR) domains, and resembles the well-conserved animal gene called ADP (antiobesity factor ADIPOSE) in fly and WDTC1 (WD40 and TPR 1) in humans. Loss of function of WDTC1 results in an increase in adipocytes, fat accumulation, and obesity. Antiadipogenic functions of WDTC1 involve regulation of fat-related gene transcription, notably through its binding to histone deacetylases (HDACs). Our sequence and phylogenetic analysis reveals that ASG2 belongs to the ADP/WDTC1 cluster. ASG2 and WDTC1 share a highly conserved organization that encompasses structural and functional motifs: seven WD40 domains and WD40 hotspot-related residues, three TPR protein-protein interaction domains, DDB1-binding elements [H-box and DWD (DDB1-binding WD40 protein)-box], and a prenylatable C-terminus. Furthermore, ASG2 involvement in fat metabolism was confirmed by reverse genetic approaches using asg2 knockout Arabidopsis plants. Under limited irradiance, asg2 mutants produce "obese" seeds characterized by increased weight, oil body density, and higher fatty acid contents. In addition, considering some ASG2- and WDTC1-peculiar properties, we show that the WDTC1 C-terminus is prenylated in vitro and HDAC-binding capability is conserved in ASG2, suggesting that the regulation mechanism and targets of ADP/WDTC1-like proteins may be conserved features. Our findings reveal the remarkable evolutionary conservation of the structure and the physiological role of ADIPOSE homologs in animals and plants.

The secret life of kinases: insights into non-catalytic signalling functions from pseudokinases.

Over the past decade, our understanding of the mechanisms by which pseudokinases, which comprise ∼10% of the human and mouse kinomes, mediate signal transduction has advanced rapidly with increasing structural, biochemical, cellular and genetic studies. Pseudokinases are the catalytically defective counterparts of conventional, active protein kinases and have been attributed functions as protein interaction domains acting variously as allosteric modulators of conventional protein kinases and other enzymes, as regulators of protein trafficking or localisation, as hubs to nucleate assembly of signalling complexes, and as transmembrane effectors of such functions. Here, by categorising mammalian pseudokinases based on their known functions, we illustrate the mechanistic diversity among these proteins, which can be viewed as a window into understanding the non-catalytic functions that can be exerted by conventional protein kinases.

Molecular cloning of two novel NPR1 homologue genes in coconut palm and analysis of their expression in response to the plant defense hormone salicylic acid

Nonexpressor of pathogenesis-related gene 1 (NPR1) codes for a transcription cofactor involved in the activation of systemic acquired resistance, a salicylic acid (SA)-dependent defense response. This work reports the cloning and characterization of two new genes, CnNPR1 and CnNPR3 from coconut, homologous to AtNPR1 of Arabidopsis thaliana. The cDNA deduced amino acid sequence contains the protein–protein interaction domains the BTB/POZ and ANKYRIN repeat domains, and a nuclear localization site (NLS). Phylogenetic analysis grouped CnNPR1 in a clade with AtNPR1 and CnNPR3 in a clade with AtNPR3, both reported genes of A. thaliana. Exogenous application of SA to coconut plantlets induced changes in the expression of CnNPR1 and CnNPR3 in leaf, stem and root tissues, providing evidence of their possible role in the signaling cascade leading to SAR in coconut palm. This is the first report on the cloning of putative key genes in the SAR-type defense mechanism in coconut palm.

The PRDM9 KRAB domain is required for meiosis and involved in protein interactions

PR domain-containing protein 9 (PRDM9) is a major regulator of the localization of meiotic recombination hotspots in the human and mouse genomes. This role involves its DNA-binding domain, which is composed of a tandem array of zinc fingers, and PRDM9-dependent trimethylation of histone H3 at lysine 4. PRDM9 is a member of the PRDM family of transcription regulators, but unlike other family members, it contains a Krüppel-associated box (KRAB)-related domain that is predicted to be a potential protein interaction domain. Here, we show that truncation of the KRAB domain of mouse PRDM9 leads to loss of PRDM9 function and altered meiotic prophase and gametogenesis. In addition, we identified proteins that interact with the KRAB domain of PRDM9 in yeast two-hybrid assay screens, particularly CXXC1, a member of the COMPASS complex. We also show that CXXC1 interacts with IHO1, an essential component of the meiotic double-strand break (DSB) machinery. As CXXC1 is orthologous to Saccharomyces cerevisiae Spp1 that links DSB sites to the DSB machinery on the chromosome axis, we propose that these molecular interactions involved in the regulation of meiotic DSB formation are conserved in mouse meiosis.

Pseudoscaffolds and anchoring proteins: the difference is in the details.

Pseudokinases and pseudophosphatases possess the ability to bind substrates without catalyzing their modification, thereby providing a mechanism to recruit potential phosphotargets away from active enzymes. Since many of these pseudoenzymes possess other characteristics such as localization signals, separate catalytic sites, and protein-protein interaction domains, they have the capacity to influence signaling dynamics in local environments. In a similar manner, the targeting of signaling enzymes to subcellular locations by A-kinase-anchoring proteins (AKAPs) allows for precise and local control of second messenger signaling events. Here, we will discuss how pseudoenzymes form 'pseudoscaffolds' and compare and contrast this compartment-specific regulatory role with the signal organization properties of AKAPs. The mitochondria will be the focus of this review, as they are dynamic organelles that influence a broad range of cellular processes such as metabolism, ATP synthesis, and apoptosis.

Purification and Crystallization of the C‐terminal Interaction Domains of TRPP Ion Channel Proteins

Members of the transient receptor potential (TRP) protein family of ion channels are essential for various cell signaling pathways throughout the body. Proteins in the polycystin subfamily of TRP channels (TRPP) form receptor‐ion channel complexes with members of the polycystic kidney disease (PKD) protein family in 3:1 stoichiometry (3TRPP:1PKD). TRPP2 forms a flow‐sensing complex in primary cilia with its PKD partners that determines left‐right asymmetry in the embryonic node and proper morphology of kidney tubules. Mutations in TRPP2 cause autosomal dominant polycystic kidney disease (ADPKD), which affects over 400,000 Americans and often leads to end stage renal failure. TRPP3, with its binding partner PKD1L3, is a sour taste receptor candidate found in taste cells in the tongue. The C‐terminus of the TRPP channel has been shown to be essential for channel assembly and function. In both TRPP2 and TRPP3, the C‐terminal coiled‐coil domains form a homotrimer and play a key role in the assembly of the TRPP/PKD complexes. Another C1 domain was found in TRPP3 that helps direct homomeric assembly. TRPP2 also has a calcium binding EF‐hand motif involved in regulation of channel activity, and an ER‐retention motif that is important for its trafficking from the ER to the cell membrane. Although the structures of the coiled‐coil domains of both proteins and the EF‐hand motif of TRPP2 have been solved separately, we still don't have an overall picture of how the different domains and motifs interact and assemble with each other. Recently, a cryo‐EM structure of TRPP2 has been reported. However, it lacks the C‐terminus. Clearly, the assembly of these proteins is multi‐faceted and significant for their function, and resolving the structure of that assembly is a necessary step towards understanding the function of the protein in vivo. Our preliminary data show oligomer formation of these proteins. In this structural study, we selected several C‐terminal fragments of all three TRPP proteins based on secondary structure prediction and functional importance, and expressed and purified them using E. coli. Our goal is to crystallize select members of the TRPP family and determine their structures through X‐ray diffraction. This study will elucidate the interaction and assembly of the C‐terminal domains of TRPP proteins and the relevance of key amino acid residues to channel function.Support or Funding InformationThis work was supported by NIH Grant DK102092.

The structure of FKBP38 in complex with the MEEVD tetratricopeptide binding-motif of Hsp90

Tetratricopeptide (TPR) domains are known protein interaction domains. We show that the TPR domain of FKBP8 selectively binds Hsp90, and interactions upstream of the conserved MEEVD motif are critical for tight binding. In contrast FKBP8 failed to bind intact Hsp70. The PPIase domain was not essential for the interaction with Hsp90 and binding was completely encompassed by the TPR domain alone. The conformation adopted by Hsp90 peptides, containing the conserved MEEVD motif, in the crystal structure were similar to that seen for the TPR domains of CHIP, AIP and Tah1. The carboxylate clamp interactions with bound Hsp90 peptide were a critical component of the interaction and mutation of Lys 307, involved in the carboxylate clamp, completely disrupted the interaction with Hsp90. FKBP8 binding to Hsp90 did not substantially influence its ATPase activity.