Conserved Distal Loop Residues in the Hsp104 and ClpB Middle Domain Contact Nucleotide-binding Domain 2 and Enable Hsp70-dependent Protein Disaggregation

The homologous hexameric AAA⁺ proteins, Hsp104 from yeast and ClpB from bacteria, collaborate with Hsp70 to dissolve disordered protein aggregates but employ distinct mechanisms of intersubunit collaboration. How Hsp104 and ClpB coordinate polypeptide handover with Hsp70 is not understood. Here, we define conserved distal loop residues between middle domain (MD) helix 1 and 2 that are unexpectedly critical for Hsp104 and ClpB collaboration with Hsp70. Surprisingly, the Hsp104 and ClpB MD distal loop does not contact Hsp70 but makes intrasubunit contacts with nucleotide-binding domain 2 (NBD2). Thus, the MD does not invariably project out into solution as in one structural model of Hsp104 and ClpB hexamers. These intrasubunit contacts as well as those between MD helix 2 and NBD1 are different in Hsp104 and ClpB. NBD2-MD contacts dampen disaggregase activity and must separate for protein disaggregation. We demonstrate that ClpB requires DnaK more stringently than Hsp104 requires Hsp70 for protein disaggregation. Thus, we reveal key differences in how Hsp104 and ClpB coordinate polypeptide handover with Hsp70, which likely reflects differential tuning for yeast and bacterial proteostasis.     

Crystal structure of PilF: functional implication in the type 4 pilus biogenesis in Pseudomonas aeruginosa

PilF is a requisite protein involved in the type 4 pilus biogenesis system from the Gram-negative human pathogenic bacteria, Pseudomonas aeruginosa. We determined the PilF structure at a 2.2A resolution; this includes six tandem tetratrico peptide repeat (TPR) units forming right-handed superhelix. PilF structure was similar to the heat shock protein organizing protein, which interacts with the C-terminal peptide of Hsp90 and Hsp70 via a concave Asn ladder in the inner groove of TPR superhelix. After simulated screening, the C-terminal pentapeptides of PilG, PilU, PilY, and PilZ proved to be a likely candidate binding to PilF, which are ones of 25 necessary components involved in the type 4 pilus biogenesis system. We proposed that PilF would be critical as a bridgehead in protein-protein interaction and thereby, PilF may bind a necessary molecule in type 4 pilus biogenesis system such as PilG, PilU, PilY, and PilZ.     

Morphometric and genetic differentiation in isolated populations of the endangered Mesoamerican stingless bee <Emphasis Type="Italic">Melipona yucatanica</Emphasis> (Hymenoptera: Apoidea) suggest the existence of a two species complex

The stingless bee Melipona yucatanica is a rare species only found in preserved forests across Mesoamerica. Morphometric and molecular analyses (DNA barcoding of the cytochrome oxidase (cox1) and microsatellites) were combined to characterize and compare populations from Mexico and Guatemala. We aim to test the hypothesis predicting that populations from these two geographic regions could be considered as distinct taxonomic units. Morphometric analyses revealed geographic differences, Guatemalan bees being larger than Mexican specimens. Bayesian analyses of the mitochondrial cox1 region and the microsatellite loci demonstrated that M. yucatanica form two clades corresponding to the Mexican and Guatemalan populations. These results suggest that M. yucatanica from Mexico and Guatemala could represent two distinct species. However, more studies are needed on their ecology and behavior to determine the possibility of gene flow between them.     

Structure of ubiquitin-fold modifier 1-specific protease UfSP2

Ubiquitin-fold modifier 1 (Ufm1)-specific protease 2 (UfSP2) is a cysteine protease that is responsible for the release of Ufm1 from Ufm1-conjugated cellular proteins, as well as for the generation of mature Ufm1 from its precursor. The 2.6 Å resolution crystal structure of mouse UfSP2 reveals that it is composed of two domains. The C-terminal catalytic domain is similar to UfSP1 with Cys²⁹⁴, Asp⁴¹⁸, His⁴²⁰, Tyr²⁸², and a regulatory loop participating in catalysis. The novel N-terminal domain shows a unique structure and plays a role in the recognition of its cellular substrate C20orf116 and thus in the recruitment of UfSP2 to the endoplasmic reticulum, where C20orf116 predominantly localizes. Mutagenesis studies were carried out to provide the structural basis for understanding the loss of catalytic activity observed in a recently identified UfSP2 mutation that is associated with an autosomal dominant form of hip dysplasia.     

Study of Circular Dichroism Modes Through Decomposition of Planar Nanostructures

We extend the plasmon hybridization method from a single nanoparticle to a complex planar nanostructure, decomposing the complex nanostructure into fundamental nanoparticle building blocks. Using gammadion nanostructure as an example, we validated the theory by comparing the field profile in the gammadion’s arms under the influence of an incident circularly polarized wave. This allows us to address the origin of the plasmonics modes in the circular dichroism (CD) spectrum. The use of this hybridization method provides a simple and intuitive explanation on how conductive and inductive coupling may result from complex planar nanostructures, allowing us to study its optical properties. Using our approach, top down hybridization studies can be applied to other complex planar structures to gain further insight on the origin of the CD modes and enhance ultrasensitive sensing of chiral micro and macro molecules.     

Image-based pooled whole-genome CRISPRi screening for subcellular phenotypes

Genome-wide CRISPR screens have transformed our ability to systematically interrogate human gene function, but are currently limited to a subset of cellular phenotypes. We report a novel pooled screening approach for a wider range of cellular and subtle subcellular phenotypes. Machine learning and convolutional neural network models are trained on the subcellular phenotype to be queried. Genome-wide screening then utilizes cells stably expressing dCas9-KRAB (CRISPRi), photoactivatable fluorescent protein (PA-mCherry), and a lentiviral guide RNA (gRNA) pool. Cells are screened by using microscopy and classified by artificial intelligence (AI) algorithms, which precisely identify the genetically altered phenotype. Cells with the phenotype of interest are photoactivated and isolated via flow cytometry, and the gRNAs are identified by sequencing. A proof-of-concept screen accurately identified PINK1 as essential for Parkin recruitment to mitochondria. A genome-wide screen identified factors mediating TFEB relocation from the nucleus to the cytosol upon prolonged starvation. Twenty-one of the 64 hits called by the neural network model were independently validated, revealing new effectors of TFEB subcellular localization. This approach, AI-photoswitchable screening (AI-PS), offers a novel screening platform capable of classifying a broad range of mammalian subcellular morphologies, an approach largely unattainable with current methodologies at genome-wide scale.     

An NMR Conformational Analysis of Cyclic Bradykinin Mimics. Evidence for a β-Turn

Abstract

A detailed NMR study is carried out in acetonitrile/water solutions on three novel cyclic bradykinin antagonist analogues, BKM-824, BKM-870, and BKM-872, to examine their solution structures, and to correlate the structures with bradykinin antagonist and anti-cancer activities. The solution structures of the cyclic peptides are correlated with the structural data for known linear bradykinin antagonists. The sequences are: BKM-824 c[Ava-Igl-Ser-DF5F-Oic- Arg] where Ava is 5-aminovaleric acid, Igl is α-(2-indanyl)glycine, F5F is pentafluorophenylalanine, and Oic is (2S,3aS,7aS)-octahydroindole-2-carboxylic acid; BKM-870; c[DArg-Arg-Add-DF5F-Oic-Arg] where Add is 12-aminododecanoic acid; and BKM-872; c[DArg-Arg-Eac-Ser-DF5F-Oic-Arg] where Eac is 6-aminocaproic acid. BKM-824 was the only peptide within this series that possessed a discernable solution structure. The NMR data indicate the presence of a type I β-turn between residues F5F⁴ and Ava¹, a C-terminal-like end. Molecular dynamics calculations show that a type I β-turn from DF5F⁴ to Ava¹ does exist although the turn was somewhat distorted. This result differs from the structures seen in linear bradykinin antagonists, which usually possess a type II II′β-turn at the C-terminal end and the presence of a defined turn is correlated with bradykinin antagonist activity. There is no solution structure for BKM-870 and BKM-872 but a correlation between the primary sequence Arg^terminal-DArg¹-Arg²-long chain aliphatic amino acid and anti-cancer activity is evident.