Mediate Protein Interactions Research Articles

The effects of histidine substitution of aromatic residues on the amyloidogenic properties of the fragment 264–277 of nucleophosmin 1

Histidine (His) plays a key role in mediating protein interactions and its unique side chain determines pH responsive self-assembling processes and thus in the formation of nanostructures. In this study, To identify novel self-assembling bioinspired sequences, we analyzed a series of peptide sequences obtained through the point mutation of aromatic residues of 264–277 fragment of nucleophosmin 1 (NPM1) with single and double histidines. Through several orthogonal biophysical techniques and under different pH and ionic strength conditions we evaluated the effects of these substitutions in the amyloidogenic features of derived peptides. The results clearly indicate that both the type of aromatic mutated residue and its position can have different effect on amyloid-like behaviors. They corroborate the crucial role exerted by Tyr271 in the self-assembling process of CTD of NPM1 in AML mutated form and add novel insights in the accurate investigation of how side chain orientations can determine successful design of innovative bioinspired materials.

Comprehensive Characterization of B-Box Zinc Finger Genes in Citrullus lanatus and Their Response to Hormone and Abiotic Stresses.

Plant B-BOX (BBX) zinc finger transcription factors play crucial roles in growth and development and the stress response. Although the BBX family has been characterized in various plants, systematic analysis in watermelon is still lacking. In this study, 25 watermelon ClBBX genes were identified. ClBBXs were grouped into five clades (Clade I, II, III, IV, and V) based on their conserved domains and phylogenetic relationships. Most of the ClBBXs (84%) might be localized in the nuclei or cytoplasm. The classification of ClBBXs was consistent with their gene structures. They were unevenly distributed in nine chromosomes except for Chr4 and Chr10, with the largest number of six members in Chr2. Segmental duplications were the major factor in ClBBX family expansion. Some BBXs of watermelon and Arabidopsis evolved from a common ancestor. In total, 254 hormonal and stress-responsive cis elements were discovered in ClBBX promoters. ClBBXs were differentially expressed in tissues, and the expression levels of ClBBX15 and 16 were higher in aboveground tissues than in roots, while the patterns of ClBBX21a, 21b, 21c, 28 and 30b were the opposite. With salicylic acid, methyl jasmonate and salt stress conditions, 17, 18 and 18 ClBBXs exhibited significant expression changes, respectively. In addition, many ClBBXs, including ClBBX29b, 30a and 30b, were also responsive to cold and osmotic stress. In summary, the simultaneous response of multiple ClBBXs to hormonal or abiotic stress suggests that they may have functional interactions in the stress hormone network. Clarifying the roles of key ClBBXs in transcriptional regulation and mediating protein interactions will be an important task. Our comprehensive characterization of the watermelon ClBBX family provides vital clues for the in-depth analysis of their biological functions in stress and hormone signaling pathways.

Crystal structure of the membrane (M) protein from a bat betacoronavirus.

The membrane (M) protein is the most abundant structural protein of coronaviruses including MERS-CoV, SARS-CoV, and SARS-CoV-2, and plays a central role in virus assembly through its interaction with various partner proteins. However, mechanistic details about how M protein interacts with others remain elusive due to lack of high-resolution structures. Here, we present the first crystal structure of a betacoronavirus M protein from Pipistrellus bat coronavirus HKU5 (batCOV5-M), which is closely related to MERS-CoV, SARS-CoV, and SARS-CoV-2 M proteins. Furthermore, an interaction analysis indicates that the carboxy-terminus of the batCOV5 nucleocapsid (N) protein mediates its interaction with batCOV5-M. Combined with a computational docking analysis an M-N interaction model is proposed, providing insight into the mechanism of M protein-mediated protein interactions.

Identification and sequencing of temperature sensitive alleles of the Anaphase Promoting Complex component mat-3 in C. elegans.

The Anaphase Promoting Complex (APC) regulates the transition from metaphase to anaphase during cell division and is important to prevent defects in chromosome segregation. In a recent temperature sensitive genetic screen looking for further genes involved in fertilization, we isolated a new temperature sensitive allele of mat-3 (as49) . We also sequenced a previously identified mat-3 ( or344 ) allele that did not previously have an annotated sequence. We determined that the as49 allele was an Alanine to Threonine (A451T) mutation in the sixth exon and the or344 mutation was a Leucine to Phenylalanine (L474F) mutation in the seventh exon. These locations of the mutant alleles are consistent with other previously annotated alleles that displayed the same metaphase to anaphase transition defect phenotype and further reinforce the importance of the tetratricopeptide repeats to mediate protein interactions.

Structural insights into the role of the WW2 domain on tandem WW–PPxY motif interactions of oxidoreductase WWOX

Class I WW domains are present in many proteins of various functions and mediate protein interactions by binding to short linear PPxY motifs. Tandem WW domains often bind peptides with multiple PPxY motifs, but the interplay of WW–peptide interactions is not always intuitive. The WW domain–containing oxidoreductase (WWOX) harbors two WW domains: an unstable WW1 capable of PPxY binding and stable WW2 that cannot bind PPxY. The WW2 domain has been suggested to act as a WW1 domain chaperone, but the underlying mechanism of its chaperone activity remains to be revealed. Here, we combined NMR, isothermal calorimetry, and structural modeling to elucidate the roles of both WW domains in WWOX binding to its PPxY-containing substrate ErbB4. Using NMR, we identified an interaction surface between these two domains that supports a WWOX conformation compatible with peptide substrate binding. Isothermal calorimetry and NMR measurements also indicated that while binding affinity to a single PPxY motif is marginally increased in the presence of WW2, affinity to a dual-motif peptide increases 10-fold. Furthermore, we found WW2 can directly bind double-motif peptides using its canonical binding site. Finally, differential binding of peptides in mutagenesis experiments was consistent with a parallel N- to C-terminal PPxY tandem motif orientation in binding to the WW1–WW2 tandem domain, validating structural models of the interaction. Taken together, our results reveal the complex nature of tandem WW-domain organization and substrate binding, highlighting the contribution of WWOX WW2 to both protein stability and target binding.

The inner side of yeast PCNA contributes to genome stability by mediating interactions with Rad18 and the replicative DNA polymerase \u03b4

PCNA is a central orchestrator of cellular processes linked to DNA metabolism. It is a binding platform for a plethora of proteins and coordinates and regulates the activity of several pathways. The outer side of PCNA comprises most of the known interacting and regulatory surfaces, whereas the residues at the inner side constitute the sliding surface facing the DNA double helix. Here, by investigating the L154A mutation found at the inner side, we show that the inner surface mediates protein interactions essential for genome stability. It forms part of the binding site of Rad18, a key regulator of DNA damage tolerance, and is required for PCNA sumoylation which prevents unscheduled recombination during replication. In addition, the L154 residue is necessary for stable complex formation between PCNA and the replicative DNA polymerase δ. Hence, its absence increases the mutation burden of yeast cells due to faulty replication. In summary, the essential role of the L154 of PCNA in guarding and maintaining stable replication and promoting DNA damage tolerance reveals a new connection between these processes and assigns a new coordinating function to the central channel of PCNA.

Immune regulator LGP2 targets Ubc13/UBE2N to mediate widespread interference with K63 polyubiquitination and NF-κB activation.

Lysine 63-linked polyubiquitin (K63-Ub) chains activate a range of cellular immune and inflammatory signaling pathways, including the mammalian antiviral response. Interferon and antiviral genes are triggered by TRAF family ubiquitin ligases that form K63-Ub chains. LGP2 is a feedback inhibitor of TRAF-mediated K63-Ub that can interfere with diverse immune signaling pathways. Our results demonstrate that LGP2 inhibits K63-Ub by association with and sequestration of the K63-Ub-conjugating enzyme, Ubc13/UBE2N. The LGP2 helicase subdomain, Hel2i, mediates protein interaction that engages and inhibits Ubc13/UBE2N, affecting control over a range of K63-Ub ligase proteins, including TRAF6, TRIM25, and RNF125, all of which are inactivated by LGP2. These findings establish a unifying mechanism for LGP2-mediated negative regulation that can modulate a variety of K63-Ub signaling pathways.

Crystal structure and molecular dynamics of human POLDIP2, a multifaceted adaptor protein in metabolism and genome stability.

Polymerase δ‐interacting protein 2 (POLDIP2, PDIP38) is a multifaceted, “moonlighting” protein, involved in binding protein partners from many different cellular processes, including mitochondrial metabolism and DNA replication and repair. How POLDIP2 interacts with many different proteins is unknown. Towards this goal, we present the crystal structure of POLDIP2 to 2.8 Å, which exhibited a compact two‐domain β‐strand‐rich globular structure, confirmed by circular dichroism and small angle X‐ray scattering approaches. POLDIP2 comprised canonical DUF525 and YccV domains, but with a conserved domain linker packed tightly, resulting in an “extended” YccV module. A central channel was observed, which we hypothesize could influence structural changes potentially mediated by redox conditions, following observation of a modified cysteine residue in the channel. Unstructured regions were rebuilt by ab initio modelling to generate a model of full‐length POLDIP2. Molecular dynamics simulations revealed a highly dynamic N‐terminal region tethered to the YccV‐domain by an extended linker, potentially facilitating interactions with distal binding partners. Models of POLDIP2 complexed with two of its partners, PrimPol and PCNA, indicated that dynamic flexibility of the POLDIP2 N‐terminus and loop regions likely mediate protein interactions.

Thousands of protein linear motif classes may still be undiscovered.

Linear motifs are short protein subsequences that mediate protein interactions. Hundreds of motif classes including thousands of motif instances are known. Our theory estimates how many motif classes remain undiscovered. As commonly done, we describe motif classes as regular expressions specifying motif length and the allowed amino acids at each motif position. We measure motif specificity for a pair of motif classes by quantifying how many motif-discriminating positions prevent a protein subsequence from matching the two classes at once. We derive theorems for the maximal number of motif classes that can simultaneously maintain a certain number of motif-discriminating positions between all pairs of classes in the motif universe, for a given amino acid alphabet. We also calculate the fraction of all protein subsequences that would belong to a motif class if all potential motif classes came into existence. Naturally occurring pairs of motif classes present most often a single motif-discriminating position. This mild specificity maximizes the potential number of coexisting motif classes, the expansion of the motif universe due to amino acid modifications and the fraction of amino acid sequences that code for a motif instance. As a result, thousands of linear motif classes may remain undiscovered.

A RanBP2-type zinc finger protein functions in intron splicing in Arabidopsis mitochondria and is involved in the biogenesis of respiratory complex I

The RanBP2 zinc finger (Znf) domain is a prevalent domain that mediates protein interaction and RNA binding. In Arabidopsis, a clade of four RanBP2 Znf-containing proteins, named the Organelle Zinc (OZ) finger family, are known or predicted to be targeted to either the mitochondria or the plastids. Previously we reported that OZ1 is absolutely required for the editing of 14 sites in chloroplasts. We now have investigated the function of OZ2, whose null mutation is embryo lethal. We rescued the null mutant by expressing wild-type OZ2 under the control of the seed-specific ABSCISIC ACID-INSENSITIVE3 (ABI3) promoter. Rescued mutant plants exhibit severely delayed development and a distinctive morphological phenotype. Genetic and biochemical analyses demonstrated that OZ2 promotes the splicing of transcripts of several mitochondrial nad genes and rps3. The splicing defect of nad transcripts results in the destabilization of complex I, which in turn affects the respiratory ability of oz2 mutants, turning on the alternative respiratory pathway, and impacting the plant development. Protein-protein interaction assays demonstrated binding of OZ2 to several known mitochondrial splicing factors targeting the same splicing events. These findings extend the known functional repertoire of the RanBP2 zinc finger domain in nuclear splicing to include plant organelle splicing.

Identifying hydrophobic protein patches to inform protein interaction interfaces

Interactions between proteins lie at the heart of numerous biological processes and are essential for the proper functioning of the cell. Although the importance of hydrophobic residues in driving protein interactions is universally accepted, a characterization of protein hydrophobicity, which informs its interactions, has remained elusive. The challenge lies in capturing the collective response of the protein hydration waters to the nanoscale chemical and topographical protein patterns, which determine protein hydrophobicity. To address this challenge, here, we employ specialized molecular simulations wherein water molecules are systematically displaced from the protein hydration shell; by identifying protein regions that relinquish their waters more readily than others, we are then able to uncover the most hydrophobic protein patches. Surprisingly, such patches contain a large fraction of polar/charged atoms and have chemical compositions that are similar to the more hydrophilic protein patches. Importantly, we also find a striking correspondence between the most hydrophobic protein patches and regions that mediate protein interactions. Our work thus establishes a computational framework for characterizing the emergent hydrophobicity of amphiphilic solutes, such as proteins, which display nanoscale heterogeneity, and for uncovering their interaction interfaces.

MicroProteins: Expanding functions and novel modes of regulation

MicroProteins: Expanding functions and novel modes of regulation

Crystal structure and mutational analysis of the human TRIM7 B30.2 domain provide insights into the molecular basis of its binding to glycogenin-1

Tripartite motif (TRIM)7 is an E3 ubiquitin ligase that was first identified through its interaction with glycogenin-1 (GN1), the autoglucosyltransferase that initiates glycogen biosynthesis. A growing body of evidence indicates that TRIM7 plays an important role in cancer development, viral pathogenesis, and atherosclerosis and, thus, represents a potential therapeutic target. TRIM family proteins share a multidomain architecture with a conserved N-terminal TRIM and a variable C-terminal domain. Human TRIM7 contains the canonical TRIM motif and a B30.2 domain at the C terminus. To contribute to the understanding of the mechanism of action of TRIM7, we solved the X-ray crystal structure of its B30.2 domain (TRIM7B30.2) in two crystal forms at resolutions of 1.6 Å and 1.8 Å. TRIM7B30.2 exhibits the typical B30.2 domain fold, consisting of two antiparallel β-sheets of seven and six strands, arranged as a distorted β-sandwich. Furthermore, two long loops partially cover the concave face of the β-sandwich defined by the β-sheet of six strands, thus forming a positively charged cavity. We used sequence conservation and mutational analyses to provide evidence of a putative binding interface for GN1. These studies showed that Leu423, Ser499, and Cys501 of TRIM7B30.2 and the C-terminal 33 amino acids of GN1 are critical for this binding interaction. Molecular dynamics simulations also revealed that hydrogen bond and hydrophobic interactions play a major role in the stability of a modeled TRIM7B30.2-GN1 C-terminal peptide complex. These data provide useful information that could be used to target this interaction for the development of potential therapeutic agents.

Edgetic perturbation signatures represent known and novel cancer biomarkers

Isoform switching is a recently characterized hallmark of cancer, and often translates to the loss or gain of domains mediating protein interactions and thus, the re-wiring of the interactome. Recent computational tools leverage domain-domain interaction data to resolve the condition-specific interaction networks from RNA-Seq data accounting for the domain content of the primary transcripts expressed. Here, we used The Cancer Genome Atlas RNA-Seq datasets to generate 642 patient-specific pairs of interactomes corresponding to both the tumor and the healthy tissues across 13 cancer types. The comparison of these interactomes provided a list of patient-specific edgetic perturbations of the interactomes associated with the cancerous state. We found that among the identified perturbations, select sets are robustly shared between patients at the multi-cancer, cancer-specific and cancer sub-type specific levels. Interestingly, the majority of the alterations do not directly involve significantly mutated genes, nevertheless, they strongly correlate with patient survival. The findings (available at EdgeExplorer: “http://webclu.bio.wzw.tum.de/EdgeExplorer”) are a new source of potential biomarkers for classifying cancer types and the proteins we identified are potential anti-cancer therapy targets.

The TRIM14 PRYSPRY domain mediates protein interaction via its basic interface.

Tripartite motif (TRIM)14 was recently shown to be an important molecule against pathogens. Its PRYSPRY domain acts as a protein-protein interaction module. TRIM14 exerts distinct functions via its PRYSPRY domain by interacting with different partners. However, the structural basis for its binding specificity remains unknown. Here we solved the crystal structure of the TRIM14 PRYSPRY domain, and found a positively charged surface that may mediate its partner specificity. Isothermal titration calorimetry reveals that the TRIM14 PRYSPRY domain binds to acidic peptides, and the analysis of the reported partners of TRIM14 is consistent with our assumption. Therefore, we demonstrate that the PRYSPRY domain of TRIM14 harbors a putative basic interface that may favorably bind to acidic amino acid residues.

Modulation of HIV-1 gene expression by binding of a ULM motif in the Rev protein to UHM-containing splicing factors.

The HIV-1 protein Rev is essential for virus replication and ensures the expression of partially spliced and unspliced transcripts. We identified a ULM (UHM ligand motif) motif in the Arginine-Rich Motif (ARM) of the Rev protein. ULMs (UHM ligand motif) mediate protein interactions during spliceosome assembly by binding to UHM (U2AF homology motifs) domains. Using NMR, biophysical methods and crystallography we show that the Rev ULM binds to the UHMs of U2AF65 and SPF45. The highly conserved Trp45 in the Rev ULM is crucial for UHM binding in vitro, for Rev co-precipitation with U2AF65 in human cells and for proper processing of HIV transcripts. Thus, Rev-ULM interactions with UHM splicing factors contribute to the regulation of HIV-1 transcript processing, also at the splicing level. The Rev ULM is an example of viral mimicry of host short linear motifs that enables the virus to interfere with the host molecular machinery.

The ICP27 Homology Domain of the Human Cytomegalovirus Protein UL69 Adopts a Dimer-of-Dimers Structure.

ABSTRACTThe UL69 protein from human cytomegalovirus (HCMV) is a multifunctional regulatory protein and a member of the ICP27 protein family conserved throughout herpesviruses. UL69 plays many roles during productive infection, including the regulation of viral gene expression, nuclear export of intronless viral RNAs, and control of host cell cycle progression. Throughout the ICP27 protein family, an ability to self-associate is correlated with the functions of these proteins in transactivating certain viral genes. Here, we determined the domain boundaries of a globular ICP27 homology domain of UL69, which mediates self-association, and characterized the oligomeric state of the isolated domain. Size exclusion chromatography coupled with multiangle light scattering (SEC-MALS) revealed that residues 200 to 540 form a stable homo-tetramer, whereas a shorter region comprising residues 248 to 536 forms a homo-dimer. Structural analysis of the UL69 tetramer by transmission electron microscopy (TEM) revealed a dimer-of-dimers three-dimensional envelope with bridge features likely from a region of the protein unique to betaherpesviruses. The data provide a structural template for tetramerization and improve our understanding of the structural diversity and features necessary for self-association within UL69 and the ICP27 family.

DNA und RNA — mehr als nur Träger der genetischen Information

DNA and RNA — the nucleic acids within our cells — are more than information carrier in the flow of genetic information within biological systems. They fulfil very diverse functions in numerous cellular processes, such as the regulation of gene expression and cell signalling, they mediate protein interactions, control enzyme activity, can catalyse chemical reactions and are crucial for cellular immunity. Here, I discuss their multifaceted roles from a nucleic acids perspective.

Alterations in the brain interactome of the intrinsically disordered N-terminal domain of the cellular prion protein (PrPC) in Alzheimer's disease.

The cellular prion protein (PrPC) is implicated in neuroprotective signaling and neurotoxic pathways in both prion diseases and Alzheimer’s disease (AD). Specifically, the intrinsically disordered N-terminal domain (N-PrP) has been shown to interact with neurotoxic ligands, such as Aβ and Scrapie prion protein (PrPSc), and to be crucial for the neuroprotective activity of PrPC. To gain further insight into cellular pathways tied to PrP, we analyzed the brain interactome of N-PrP. As a novel approach employing recombinantly expressed PrP and intein-mediated protein ligation, we used N-PrP covalently coupled to beads as a bait for affinity purification. N-PrP beads were incubated with human AD or control brain lysates. N-PrP binding partners were then identified by electrospray ionization tandem mass spectrometry (nano ESI-MS/MS). In addition to newly identified proteins we found many previously described PrP interactors, indicating a crucial role of the intrinsically disordered part of PrP in mediating protein interactions. Moreover, some interactors were found only in either non-AD or AD brain, suggesting aberrant PrPC interactions in the pathogenesis of AD.

Structure of frequency-interacting RNA helicase from Neurospora crassa reveals high flexibility in a domain critical for circadian rhythm and RNA surveillance.

The FRH (frequency-interacting RNA helicase) protein is the Neurospora crassa homolog of yeast Mtr4, an essential RNA helicase that plays a central role in RNA metabolism as an activator of the nuclear RNA exosome. FRH is also a required component of the circadian clock, mediating protein interactions that result in the rhythmic repression of gene expression. Here we show that FRH unwinds RNA substrates in vitro with a kinetic profile similar to Mtr4, indicating that while FRH has acquired additional functionality, its core helicase function remains intact. In contrast with the earlier FRH structures, a new crystal form of FRH results in an ATP binding site that is undisturbed by crystal contacts and adopts a conformation consistent with nucleotide binding and hydrolysis. Strikingly, this new FRH structure adopts an arch domain conformation that is dramatically altered from previous structures. Comparison of the existing FRH structures reveals conserved hinge points that appear to facilitate arch motion. Regions in the arch have been previously shown to mediate a variety of protein-protein interactions critical for RNA surveillance and circadian clock functions. The conformational changes highlighted in the FRH structures provide a platform for investigating the relationship between arch dynamics and Mtr4/FRH function.