Human Hsp70 Substrate-Binding Domains Recognize Distinct Client Proteins.

The 13 Hsp70 proteins in humans act on unique sets of substrates with diversity often being attributed to J-domain-containing protein (Hsp40 or JDP) cofactors. We were therefore surprised to find drastically different binding affinities for Hsp70-peptide substrates, leading us to probe substrate specificity among the 8 canonical Hsp70s from humans. We used peptide arrays to characterize Hsp70 binding and then mined these data using machine learning to develop an algorithm for isoform-specific prediction of Hsp70 binding sequences. The results of this algorithm revealed recognition patterns not predicted based on local sequence alignments. We then showed that none of the human isoforms can complement heat-shocked DnaK knockout Escherichia coli cells. However, chimeric Hsp70s consisting of the human nucleotide-binding domain and the substrate-binding domain of DnaK complement during heat shock, providing further evidence in vivo of the divergent function of the Hsp70 substrate-binding domains. We also demonstrated that the differences in heat shock complementation among the chimeras are not due to loss of DnaJ binding. Although we do not exclude JDPs as additional specificity factors, our data demonstrate substrate specificity among the Hsp70s, which has important implications for inhibitor development in cancer and neurodegeneration.

The run-on oligomer filament enzyme mechanism of SgrAI: Part 1. Assembly kinetics of the run-on oligomer filament.

Filament or run-on oligomer formation by metabolic enzymes is now recognized as a widespread phenomenon having potentially unique enzyme regulatory properties and biological roles, and its dysfunction is implicated in human diseases such as cancer, diabetes, and developmental disorders. SgrAI is a bacterial allosteric type II restriction endonuclease that binds to invading phage DNA, may protect the host DNA from off-target cleavage activity, and forms run-on oligomeric filaments with enhanced DNA-cleavage activity and altered DNA sequence specificity. However, the mechanisms of SgrAI filament growth, cooperativity in filament formation, sequestration of enzyme activity, and advantages over other filament mechanisms remain unknown. In this first of a two-part series, we developed methods and models to derive association and dissociation rate constants of DNA-bound SgrAI in run-on oligomers and addressed the specific questions of cooperativity and filament growth mechanisms. We show that the derived rate constants are consistent with the run-on oligomer sizes determined by EM analysis and are most consistent with a noncooperative growth mode of the run-on oligomer. These models and methods are extended in the accompanying article to include the full DNA-cleavage pathway and address specific questions related to the run-on oligomer mechanism including the sequestration of DNA-cleavage activity and trapping of products.

DNA Binding and Cleavage by the Human Parvovirus B19 NS1 Nuclease Domain.

Infection with human parvovirus B19 (B19V) has been associated with a myriad of illnesses, including erythema infectiosum (Fifth disease), hydrops fetalis, arthropathy, hepatitis, and cardiomyopathy, and also possibly the triggering of any number of different autoimmune diseases. B19V NS1 is a multidomain protein that plays a critical role in viral replication, with predicted nuclease, helicase, and gene transactivation activities. Herein, we investigate the biochemical activities of the nuclease domain (residues 2-176) of B19V NS1 (NS1-nuc) in sequence-specific DNA binding of the viral origin of replication sequences, as well as those of promoter sequences, including the viral p6 and the human p21, TNFα, and IL-6 promoters previously identified in NS1-dependent transcriptional transactivation. NS1-nuc was found to bind with high cooperativity and with multiple (five to seven) copies to the NS1 binding elements (NSBE) found in the viral origin of replication and the overlapping viral p6 promoter DNA sequence. NS1-nuc was also found to bind cooperatively with at least three copies to the GC-rich Sp1 binding sites of the human p21 gene promoter. Only weak or nonspecific binding of NS1-nuc to the segments of the TNFα and IL-6 promoters was found. Cleavage of DNA by NS1-nuc occurred at the expected viral sequence (the terminal resolution site), but only in single-stranded DNA, and NS1-nuc was found to covalently attach to the 5' end of the DNA at the cleavage site. Off-target cleavage by NS1-nuc was also identified.