scholarly journals Picornaviral polymerase domain exchanges reveal a modular basis for distinct biochemical activities of viral RNA-dependent RNA polymerases

2020 ◽  
Vol 295 (31) ◽  
pp. 10624-10637 ◽  
Author(s):  
Colleen L. Watkins ◽  
Brian J. Kempf ◽  
Stéphanie Beaucourt ◽  
David J. Barton ◽  
Olve B. Peersen
Keyword(s):  
Protein Interactions ◽  
Active Sites ◽  
Specific Protein ◽  
Rna Polymerases ◽  
Viral Fitness ◽  
Structural Basis ◽  
Protein Protein Interactions ◽  
Elongation Complex ◽  
Domain Interactions ◽  
Species Specific

Picornaviral RNA-dependent RNA polymerases (RdRPs) have low replication fidelity that is essential for viral fitness and evolution. Their global fold consists of the classical “cupped right hand” structure with palm, fingers, and thumb domains, and these RdRPs also possess a unique contact between the fingers and thumb domains. This interaction restricts movements of the fingers, and RdRPs use a subtle conformational change within the palm domain to close their active sites for catalysis. We have previously shown that this core RdRP structure and mechanism provide a platform for polymerases to fine-tune replication rates and fidelity to optimize virus fitness. Here, we further elucidated the structural basis for differences in replication rates and fidelity among different viruses by generating chimeric RdRPs from poliovirus and coxsackievirus B3. We designed these chimeric polymerases by exchanging the fingers, pinky finger, or thumb domains. The results of biochemical, rapid-quench, and stopped-flow assays revealed that differences in biochemical activity map to individual modular domains of this polymerase. We found that the pinky finger subdomain is a major regulator of initiation and that the palm domain is the major determinant of catalytic rate and nucleotide discrimination. We further noted that thumb domain interactions with product RNA regulate translocation and that the palm and thumb domains coordinately control elongation complex stability. Several RdRP chimeras supported the growth of infectious poliovirus, providing insights into enterovirus species–specific protein–protein interactions required for virus replication.

scholarly journals Molecular and structural mechanisms of ZZ domain-mediated cargo recognition by autophagy receptor Nbr1

2021 ◽  
Author(s):  
Ying-Ying Wang ◽  
Jianxiu Zhang ◽  
Xiao-Man Liu ◽  
Meng-Qiu Dong ◽  
Keqiong Ye ◽  
...  
Keyword(s):  
High Resolution ◽  
Fission Yeast ◽  
Protein Interactions ◽  
Specific Protein ◽  
Structural Basis ◽  
Protein Protein Interactions ◽  
Selective Autophagy ◽  
Specific Manner ◽  
Autophagy Pathway ◽  
Structural Mechanisms

AbstractIn selective autophagy, cargo selectivity is determined by autophagy receptors. However, it remains scarcely understood how autophagy receptors recognize specific protein cargos. In the fission yeast Schizosaccharomyces pombe, a selective autophagy pathway termed Nbr1-mediated vacuolar targeting (NVT) employs Nbr1, an autophagy receptor conserved across eukaryotes including humans, to target cytosolic hydrolases into the vacuole. Here, we identify two new NVT cargos, the mannosidase Ams1 and the aminopeptidase Ape4, that bind competitively to the first ZZ domain of Nbr1 (Nbr1-ZZ1). High-resolution cryo-EM analyses reveal how a single ZZ domain recognizes two distinct protein cargos. Nbr1-ZZ1 not only recognizes the N-termini of cargos via a conserved acidic pocket, similar to other characterized ZZ domains, but also engages additional parts of cargos in a cargo-specific manner. Our findings unveil a single-domain bispecific mechanism of autophagy cargo recognition, elucidate its underlying structural basis, and expand the understanding of ZZ domain-mediated protein-protein interactions.

scholarly journals Structural Profiling of Bacterial Effectors Reveals Enrichment of Host-Interacting Domains and Motifs

2021 ◽  
Vol 8 ◽  
Author(s):  
Yangchun Frank Chen ◽  
Yu Xia
Keyword(s):  
Protein Interactions ◽  
Effector Proteins ◽  
Host Cells ◽  
Structural Basis ◽  
Protein Protein Interactions ◽  
Specific Domain ◽  
Bacterial Proteins ◽  
Domain Interactions ◽  
Host Signaling ◽  
Bacterial Effectors

Effector proteins are bacterial virulence factors secreted directly into host cells and, through extensive interactions with host proteins, rewire host signaling pathways to the advantage of the pathogen. Despite the crucial role of globular domains as mediators of protein-protein interactions (PPIs), previous structural studies of bacterial effectors are primarily focused on individual domains, rather than domain-mediated PPIs, which limits their ability to uncover systems-level molecular recognition principles governing host-bacteria interactions. Here, we took an interaction-centric approach and systematically examined the potential of structural components within bacterial proteins to engage in or target eukaryote-specific domain-domain interactions (DDIs). Our results indicate that: 1) effectors are about six times as likely as non-effectors to contain host-like domains that mediate DDIs exclusively in eukaryotes; 2) the average domain in effectors is about seven times as likely as that in non-effectors to co-occur with DDI partners in eukaryotes rather than in bacteria; and 3) effectors are about nine times as likely as non-effectors to contain bacteria-exclusive domains that target host domains mediating DDIs exclusively in eukaryotes. Moreover, in the absence of host-like domains or among pathogen proteins without domain assignment, effectors harbor a higher variety and density of short linear motifs targeting host domains that mediate DDIs exclusively in eukaryotes. Our study lends novel quantitative insight into the structural basis of effector-induced perturbation of host-endogenous PPIs and may aid in the design of selective inhibitors of host-pathogen interactions.

scholarly journals Structural basis of the XPB–Bax1 complex as a dynamic helicase–nuclease machinery for DNA repair

2020 ◽  
Vol 48 (11) ◽  
pp. 6326-6339 ◽  
Author(s):  
Kevin DuPrez ◽  
Feng He ◽  
Zhenhang Chen ◽  
Eduardo Hilario ◽  
Li Fan
Keyword(s):  
Dna Repair ◽  
Protein Interactions ◽  
Active Sites ◽  
Atp Hydrolysis ◽  
Excision Repair ◽  
Dna Lesions ◽  
Structural Basis ◽  
Protein Protein Interactions ◽  
Nucleotide Excision ◽  
First Time

Abstract Nucleotide excision repair (NER) is a major DNA repair pathway for a variety of DNA lesions. XPB plays a key role in DNA opening at damage sites and coordinating damage incision by nucleases. XPB is conserved from archaea to human. In archaea, XPB is associated with a nuclease Bax1. Here we report crystal structures of XPB in complex with Bax1 from Archaeoglobus fulgidus (Af) and Sulfolobus tokodaii (St). These structures reveal for the first time four domains in Bax1, which interacts with XPB mainly through its N-terminal domain. A Cas2-like domain likely helps to position Bax1 at the forked DNA allowing the nuclease domain to incise one arm of the fork. Bax1 exists in monomer or homodimer but forms a heterodimer exclusively with XPB. StBax1 keeps StXPB in a closed conformation and stimulates ATP hydrolysis by XPB while AfBax1 maintains AfXPB in the open conformation and reduces its ATPase activity. Bax1 contains two distinguished nuclease active sites to presumably incise DNA damage. Our results demonstrate that protein-protein interactions regulate the activities of XPB ATPase and Bax1 nuclease. These structures provide a platform to understand the XPB-nuclease interactions important for the coordination of DNA unwinding and damage incision in eukaryotic NER.

scholarly journals Structure ofPlasmodium falciparumorotate phosphoribosyltransferase with autologous inhibitory protein–protein interactions

2015 ◽  
Vol 71 (5) ◽  
pp. 600-608 ◽  
Author(s):  
Shiva Kumar ◽  
Kalyanaraman Krishnamoorthy ◽  
Devaraja G. Mudeppa ◽  
Pradipsinh K. Rathod
Keyword(s):  
Protein Interactions ◽  
Active Sites ◽  
De Novo ◽  
Low Complexity ◽  
Specific Protein ◽  
Severe Form ◽  
Protein Protein Interactions ◽  
Inhibitory Protein ◽  
Pyrimidine Synthesis ◽  
Salvage Pathways

The most severe form of malaria is caused by the obligate parasitePlasmodium falciparum. Orotate phosphoribosyltransferase (OPRTase) is the fifth enzyme in thede novopyrimidine-synthesis pathway in the parasite, which lacks salvage pathways. Among all of the malariade novopyrimidine-biosynthesis enzymes, the structure ofP. falciparumOPRTase (PfOPRTase) was the only one unavailable until now.PfOPRTase that could be crystallized was obtained after some low-complexity sequences were removed. Four catalytic dimers were seen in the asymmetic unit (a total of eight polypeptides). In addition to revealing unique amino acids in thePfOPRTase active sites, asymmetric dimers in the larger structure pointed to novel parasite-specific protein–protein interactions that occlude the catalytic active sites. The latter could potentially modulatePfOPRTase activity in parasites and possibly provide new insights for blockingPfOPRTase functions.

scholarly journals Short loop motif profiling of protein interaction networks in acute myeloid leukaemia

2018 ◽  
Author(s):  
Sun Sook Chung ◽  
Anna Laddach ◽  
N. Shaun B. Thomas ◽  
Franca Fraternali
Keyword(s):  
Acute Myeloid Leukaemia ◽  
Myeloid Leukaemia ◽  
Protein Interactions ◽  
Active Sites ◽  
Specific Protein ◽  
Protein Protein Interactions ◽  
Ppi Networks ◽  
Short Loop ◽  
Genomics And Proteomics ◽  
Acute Myeloid

AbstractRecent advances in biotechnologies for genomics and proteomics have expanded our understanding of biological components which play crucial roles in complex mechanisms related to cancer. However, it is still challenging to extract from the available knowledge reliable targets to use in a translational setting. The reasons for this are manifold, but essentially distilling real biological signal from heterogeneous “big data” collections is the major hurdle. Here, we aim to establish an in-silico pipeline to explore mutations and their effects on protein-protein interactions, with a focus on acute myeloid leukaemia (AML), one of the most common blood cancers with the highest mortality rate. Our method, based on cyclic interactions of a small number of proteins topologically linked in the network (short loop network motifs), highlights specific protein-protein interactions (PPIs) and their functions in AML when compared with other leukaemias. We also developed a new property named ‘short loop commonality’ to measure indirect PPIs occurring via common short loop interactions. This new method detects “modules” of PPI networks (PPINs) enriched with common biological functions which have proteins that contain mutation hotspots. We further perform 3D structural modelling to extract atomistic details, which shows that such hotspots map to PPI interfaces as well as active sites. Thus, our study proposes a framework for the macroscopic and microscopic investigation of PPINs, their relation to cancers, and highlights important functional modules in the network to be exploited in targeted drug screening.

Structure, Function, Involvement in Diseases and Targeting of 14-3-3 Proteins: An Update

2018 ◽  
Vol 25 (1) ◽  
pp. 5-21 ◽  
Author(s):  
Ylenia Cau ◽  
Daniela Valensin ◽  
Mattia Mori ◽  
Sara Draghi ◽  
Maurizio Botta
Keyword(s):  
Protein Interactions ◽  
Molecular Mechanisms ◽  
Physiological Role ◽  
Specific Protein ◽  
Protein Protein Interactions ◽  
Cellular Processes ◽  
Protein Partners ◽  
High Sequence Homology ◽  
Small Molecule Modulators

14-3-3 is a class of proteins able to interact with a multitude of targets by establishing protein-protein interactions (PPIs). They are usually found in all eukaryotes with a conserved secondary structure and high sequence homology among species. 14-3-3 proteins are involved in many physiological and pathological cellular processes either by triggering or interfering with the activity of specific protein partners. In the last years, the scientific community has collected many evidences on the role played by seven human 14-3-3 isoforms in cancer or neurodegenerative diseases. Indeed, these proteins regulate the molecular mechanisms associated to these diseases by interacting with (i) oncogenic and (ii) pro-apoptotic proteins and (iii) with proteins involved in Parkinson and Alzheimer diseases. The discovery of small molecule modulators of 14-3-3 PPIs could facilitate complete understanding of the physiological role of these proteins, and might offer valuable therapeutic approaches for these critical pathological states.

Metallocluster transactions: dynamic protein interactions guide the biosynthesis of Fe–S clusters in bacteria

2018 ◽  
Vol 46 (6) ◽  
pp. 1593-1603 ◽  
Author(s):  
Chenkang Zheng ◽  
Patricia C. Dos Santos
Keyword(s):  
Protein Interactions ◽  
Protein Complexes ◽  
Specific Protein ◽  
Biological Synthesis ◽  
Cluster Assembly ◽  
Sequence Motifs ◽  
Protein Protein Interactions ◽  
Cysteine Desulfurase ◽  
Wide Range ◽  
Domains Of Life

Iron–sulfur (Fe–S) clusters are ubiquitous cofactors present in all domains of life. The chemistries catalyzed by these inorganic cofactors are diverse and their associated enzymes are involved in many cellular processes. Despite the wide range of structures reported for Fe–S clusters inserted into proteins, the biological synthesis of all Fe–S clusters starts with the assembly of simple units of 2Fe–2S and 4Fe–4S clusters. Several systems have been associated with the formation of Fe–S clusters in bacteria with varying phylogenetic origins and number of biosynthetic and regulatory components. All systems, however, construct Fe–S clusters through a similar biosynthetic scheme involving three main steps: (1) sulfur activation by a cysteine desulfurase, (2) cluster assembly by a scaffold protein, and (3) guided delivery of Fe–S units to either final acceptors or biosynthetic enzymes involved in the formation of complex metalloclusters. Another unifying feature on the biological formation of Fe–S clusters in bacteria is that these systems are tightly regulated by a network of protein interactions. Thus, the formation of transient protein complexes among biosynthetic components allows for the direct transfer of reactive sulfur and Fe–S intermediates preventing oxygen damage and reactions with non-physiological targets. Recent studies revealed the importance of reciprocal signature sequence motifs that enable specific protein–protein interactions and consequently guide the transactions between physiological donors and acceptors. Such findings provide insights into strategies used by bacteria to regulate the flow of reactive intermediates and provide protein barcodes to uncover yet-unidentified cellular components involved in Fe–S metabolism.

scholarly journals Bi-directional protein-protein interactions control liquid-liquid phase separation of PSD-95 and its interaction partners

2021 ◽  
Author(s):  
Nikolaj Riis Christensen ◽  
Christian Parsbæk Pedersen ◽  
Vita Sereikaite ◽  
Jannik Nedergaard Pedersen ◽  
Maria Vistrup-Parry ◽  
...  
Keyword(s):  
Phase Separation ◽  
Liquid Phase ◽  
Protein Interactions ◽  
Postsynaptic Density ◽  
Specific Protein ◽  
Liquid Phase Separation ◽  
Protein Protein Interactions ◽  
New Perspective ◽  
Liquid Liquid Phase Separation

SUMMARYThe organization of the postsynaptic density (PSD), a protein-dense semi-membraneless organelle, is mediated by numerous specific protein-protein interactions (PPIs) which constitute a functional post-synapse. Postsynaptic density protein 95 (PSD-95) interacts with a manifold of proteins, including the C-terminal of transmembrane AMPA receptor (AMAPR) regulatory proteins (TARPs). Here, we uncover the minimal essential peptide responsible for the stargazin (TARP-γ2) mediated liquid-liquid phase separation (LLPS) formation of PSD-95 and other key protein constituents of the PSD. Furthermore, we find that pharmacological inhibitors of PSD-95 can facilitate formation of LLPS. We found that in some cases LLPS formation is dependent on multivalent interactions while in other cases short peptides carrying a high charge are sufficient to promote LLPS in complex systems. This study offers a new perspective on PSD-95 interactions and their role in LLPS formation, while also considering the role of affinity over multivalency in LLPS systems.

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