Condensins are multisubunit protein complexes that play a fundamental role in the structural and functional organization of chromosomes in the three domains of life. Most eukaryotic species have two different types of condensin complexes, known as condensins I and II, that fulfill nonoverlapping functions and are subjected to differential regulation during mitosis and meiosis. Recent studies revealed that the two complexes contribute to a wide variety of interphase chromosome functions, such as gene regulation, recombination, and repair. Also emerging are their cell type- and tissue-specific functions and relevance to human disease. Biochemical and structural analyses of eukaryotic and bacterial condensins steadily uncover the mechanisms of action of this class of highly sophisticated molecular machines. Future studies on condensins will not only enhance our understanding of chromosome architecture and dynamics, but also help address a previously underappreciated yet profound set of questions in chromosome biology.

http://genesdev.cshlp.org/content/26/15/1659.full.pdf

Condensins: universal organizers of chromosomes with diverse functions

Chemogenetic characterization through in vitro evolution combined with whole-genome analysis can identify antimalarial drug targets and drug-resistance genes. We performed a genome analysis of 262 Plasmodium falciparum parasites resistant to 37 diverse compounds. We found 159 gene amplifications and 148 nonsynonymous changes in 83 genes associated with drug-resistance acquisition, where gene amplifications contributed to one-third of resistance acquisition events. Beyond confirming previously identified multidrug-resistance mechanisms, we discovered hitherto unrecognized drug target-inhibitor pairs, including thymidylate synthase and a benzoquinazolinone, farnesyltransferase and a pyrimidinedione, and a dipeptidylpeptidase and an arylurea. This exploration of the P. falciparum resistome and druggable genome will likely guide drug discovery and structural biology efforts, while also advancing our understanding of resistance mechanisms available to the malaria parasite.

/pdf/mapping-the-malaria-parasite-druggable-genome-by-using-in-4fxp33wbqp.pdf

Mapping the malaria parasite druggable genome by using in vitro evolution and chemogenomics

The class Gammaproteobacteria, which forms one of the largest groups within bacteria, is currently distinguished from other bacteria solely on the basis of its branching in phylogenetic trees. No molecular or biochemical characteristic is known that is unique to the class Gammaproteobacteria or its different subgroups (orders). The relationship among different orders of gammaproteobacteria is also not clear. In this study, we present detailed phylogenomic and comparative genomic analyses on gammaproteobacteria that clarify some of these issues. Phylogenetic trees based on concatenated sequences for 13 and 36 universally distributed proteins were constructed for 45 members of the class Gammaproteobacteria covering 13 of its 14 orders. In these trees, species from a number of the subgroups formed distinct clades and their relative branching order was indicated as follows (from the most recent to the earliest diverging): Enterobacteriales >Pasteurellales >Vibrionales, Aeromonadales >Alteromonadales >Oceanospirillales, Pseudomonadales >Chromatiales, Legionellales, Methylococcales, Xanthomonadales, Cardiobacteriales, Thiotrichales. Four conserved indels in four widely distributed proteins that are specific for gammaproteobacteria are also described. A 2 aa deletion in 5′-phosphoribosyl-5-aminoimidazole-4-carboxamide transformylase (AICAR transformylase; PurH) was a distinctive characteristic of all gammaproteobacteria (except Francisella tularensis). Two other conserved indels (a 4 aa deletion in RNA polymerase β-subunit and a 1 aa deletion in ribosomal protein L16) were found uniquely in various species of the orders Enterobacteriales, Pasteurellales, Vibrionales, Aeromonadales and Alteromonadales, but were not found in other gammaproteobacteria. Lastly, a 2 aa deletion in leucyl-tRNA synthetase was commonly present in the above orders of the class Gammaproteobacteria and also in some members of the order Oceanospirillales. The presence of the conserved indels in these gammaproteobacterial orders indicates that species from these orders shared a common ancestor that was separate from other bacteria, a suggestion that is supported by phylogenetic studies. Systematic blastp searches were also conducted on various open reading frames (ORFs) in the genome of Escherichia coli K-12. These analyses identified 75 proteins that were unique to most members of the class Gammaproteobacteria or were restricted to species from some of its main orders (Enterobacteriales; Enterobacteriales and Pasteurellales; Enterobacteriales, Pasteurellales, Vibrionales, Aeromonadales and Alteromonadales; and the Enterobacteriales,
Pasteurellales, Vibrionales, Aeromonadales, Alteromonadales, Oceanospirillales and Pseudomonadales etc.). The genes for these proteins have evolved at various stages during the evolution of gammaproteobacteria and their species distribution pattern, in conjunction with other results presented here, provide valuable information regarding the evolutionary relationships among these bacteria.

/pdf/phylogenomics-and-protein-signatures-elucidating-the-3xrtm4fsvo.pdf

Phylogenomics and protein signatures elucidating the evolutionary relationships among the Gammaproteobacteria

In the current work, unanticipated synthetic byproducts were obtained arising from alkylation of the δ1 nitrogen (N3) of the histidine imidazole ring of the polo-like kinase-1 (Plk1) polo-box domain (PBD)-binding peptide PLHSpT. For the highest affinity byproduct, bearing a C6H5(CH2)8– group, a Plk1 PBD co-crystal structure revealed a new binding channel that had previously been occluded. An N-terminal PEGylated version of this peptide containing a hydrolytically-stable phosphothreonyl residue (pT) bound to the Plk1 PBD with affinity equal to the non-PEGylated parent, yet it exhibited significantly less interaction with the PBDs of the two closely-related Plk2 or Plk3. Treatment of cultured cells with this PEGylated peptide resulted in Plk1 delocalization from centrosomes and kinetochores, and chromosome misalignment that effectively induced mitotic block and apoptotic cell death. This work provides new insights that may advance efforts to develop Plk1 PBD-binding inhibitors as potential Plk1-specific anticancer therapeutic agents.

Serendipitous alkylation of a Plk1 ligand uncovers a new binding channel

Bacteria and archaea possess several different SMC-like proteins, which perform essential functions in a variety of chromosome dynamics, such as chromosome compaction, segregation, and DNA repair. SMC-like proteins localize to distinct sites within the cells at different time points in the cell cycle, or are recruited to sites of DNA breaks and damage. The bacterial SMC (MukB) complex appears to perform a condensin-like function, while SbcC and RecN act early during DNA repair, but apparently at different sites within the cells. Thus, bacterial SMC-like proteins have dynamic functions in chromosome segregation and maintenance of genetic stability.

Dynamics of the bacterial SMC complex and SMC-like proteins involved in DNA repair.

Forkhead-associated (FHA) domains are small phosphopeptide recognition modules found in eubacterial and eukaryotic, but not archeal, genomes. Although they were originally found in forkhead-type transcription factors, they have now been identified in many other signaling proteins. FHA domains share a remarkably conserved fold despite very low sequence conservation. They only have five conserved amino acids that are important for binding to phosphorylated epitopes. Recent work from several laboratories has demonstrated that FHA domains can mediate many interactions that do not depend on their ability to recognize a phosphorylated threonine. In this review, we present structural and biochemical work that has unveiled novel interaction interfaces on FHA domains. We discuss how these non-canonical interactions modulate the recognition of phosphorylated and non-phosphorylated substrates, as well as protein oligomerization - events that collectively determine FHA function.

FHA domains: Phosphopeptide binding and beyond

Together with cyclin-dependent kinases, the Dbf4-dependent kinase (DDK) is essential to activate the Mcm2-7 helicase and, hence, initiate DNA replication in eukaryotes. Beyond its role as the regulatory subunit of the DDK complex, the Dbf4 protein also regulates the activity of other cell cycle kinases to mediate the checkpoint response and prevent premature mitotic exit under stress. Two features that are unusual in DNA replication proteins characterize Dbf4. The first is its evolutionary divergence; the second is how its conserved motifs are combined to form distinct functional units. This structural plasticity appears to be at odds with the conserved functions of Dbf4. In this review, we summarize recent genetic, biochemical and structural work delineating the multiple interactions mediated by Dbf4 and its various functions during the cell cycle. We also discuss how the limited sequence conservation of Dbf4 may be an advantage to regulate the activities of multiple cell cycle kinases.

https://www.tandfonline.com/doi/pdf/10.4161/cc.24416

Dbf4: the whole is greater than the sum of its parts.

Saccharomyces cerevisiae Dbf4 has unique fold necessary for interaction with Rad53 kinase.

A novel non-canonical forkhead-associated (FHA) domain-binding interface mediates the interaction between Rad53 and Dbf4 proteins

Forkhead-associated (FHA) domains are phosphopeptide recognition modules found in many signaling proteins. The Saccharomyces cerevisiae protein kinase Rad53 is a key regulator of the DNA damage checkpoint and uses its two FHA domains to interact with multiple binding partners during the checkpoint response. One of these binding partners is the Dbf4-dependent kinase (DDK), a heterodimer composed of the Cdc7 kinase and its regulatory subunit Dbf4. Binding of Rad53 to DDK, through its N-terminal FHA (FHA1) domain, ultimately inhibits DDK kinase activity, thereby preventing firing of late origins. We have previously found that the FHA1 domain of Rad53 binds simultaneously to Dbf4 and a phosphoepitope, suggesting that this domain functions as an ‘AND’ logic gate. Here, we present the crystal structures of the FHA1 domain of Rad53 bound to Dbf4, in the presence and absence of a Cdc7 phosphorylated peptide. Our results reveal how the FHA1 uses a canonical binding interface to recognize the Cdc7 phosphopeptide and a non-canonical interface to bind Dbf4. Based on these data we propose a mechanism to explain how Rad53 enhances the specificity of FHA1-mediated transient interactions.

/pdf/and-logic-gates-at-work-crystal-structure-of-rad53-bound-to-4vuejugb4f.pdf

Lindsay A. Matthews

Papers

FHA domains: Phosphopeptide binding and beyond

Dbf4: the whole is greater than the sum of its parts.

Saccharomyces cerevisiae Dbf4 has unique fold necessary for interaction with Rad53 kinase.

A novel non-canonical forkhead-associated (FHA) domain-binding interface mediates the interaction between Rad53 and Dbf4 proteins

'AND' logic gates at work: Crystal structure of Rad53 bound to Dbf4 and Cdc7.