Morgan E. DeSantis

The elusive middle domain of Hsp104 and ClpB: location and function

Hsp104 in yeast and ClpB in bacteria are homologous, hexameric AAA+ proteins and Hsp100 chaperones, which function in the stress response as ring-translocases that drive protein disaggregation and reactivation. Both Hsp104 and ClpB contain a distinctive coiled-coil middle domain (MD) inserted in the first AAA+ domain, which distinguishes them from other AAA+ proteins and Hsp100 family members. Here, we focus on recent developments concerning the location and function of the MD in these hexameric molecular machines, which remains an outstanding question. While the atomic structure of the hexameric assembly of Hsp104 and ClpB remains uncertain, recent advances have illuminated that the MD is critical for the intrinsic disaggregase activity of the hexamer and mediates key functional interactions with the Hsp70 chaperone system (Hsp70 and Hsp40) that empower protein disaggregation.

Fission Yeast Does Not Age under Favorable Conditions, but Does So after Stress.

BACKGROUND Many unicellular organisms age: as time passes, they divide more slowly and ultimately die. In budding yeast, asymmetric segregation of cellular damage results in aging mother cells and rejuvenated daughters. We hypothesize that the organisms in which this asymmetry is lacking, or can be modulated, may not undergo aging. RESULTS We performed a complete pedigree analysis of microcolonies of the fission yeast Schizosaccharomyces pombe growing from a single cell. When cells were grown under favorable conditions, none of the lineages exhibited aging, which is defined as a consecutive increase in division time and increased death probability. Under favorable conditions, few cells died, and their death was random and sudden rather than following a gradual increase in division time. Cell death correlated with the inheritance of Hsp104-associated protein aggregates. After stress, the cells that inherited large aggregates aged, showing a consecutive increase in division time and an increased death probability. Their sisters, who inherited little or no aggregates, did not age. CONCLUSIONS We conclude that S. pombe does not age under favorable growth conditions, but does so under stress. This transition appears to be passive rather than active and results from the formation of a single large aggregate, which segregates asymmetrically at the subsequent cell division. We argue that this damage-induced asymmetric segregation has evolved to sacrifice some cells so that others may survive unscathed after severe environmental stresses.

The Hsp104 N-terminal domain enables disaggregase plasticity and potentiation.

The structural basis by which Hsp104 dissolves disordered aggregates and prions is unknown. A single subunit within the Hsp104 hexamer can solubilize disordered aggregates, whereas prion dissolution requires collaboration by multiple Hsp104 subunits. Here, we establish that the poorly understood Hsp104 N-terminal domain (NTD) enables this operational plasticity. Hsp104 lacking the NTD (Hsp104(ΔN)) dissolves disordered aggregates but cannot dissolve prions or be potentiated by activating mutations. We define how Hsp104(ΔN) invariably stimulates Sup35 prionogenesis by fragmenting prions without solubilizing Sup35, whereas Hsp104 couples Sup35 prion fragmentation and dissolution. Volumetric reconstruction of Hsp104 hexamers in ATPγS, ADP-AlFx (hydrolysis transition state mimic), and ADP via small-angle X-ray scattering revealed a peristaltic pumping motion upon ATP hydrolysis, which drives directional substrate translocation through the central Hsp104 channel and is profoundly altered in Hsp104(ΔN). We establish that the Hsp104 NTD enables cooperative substrate translocation, which is critical for prion dissolution and potentiated disaggregase activity.

Association Energetics of Cross-Reactive and Specific Antibodies†

HyHEL-8, HyHEL-10, and HyHEL-26 (HH8, HH10, and HH26, respectively) are murine monoclonal IgG(1) antibodies which share over 90% variable-region amino acid sequence identity and recognize identical structurally characterized epitopes on hen egg white lysozyme (HEL). Previous immunochemical and surface plasmon resonance-based studies have shown that these antibodies differ widely in their tolerance of mutations in the epitope. While HH8 is the most cross-reactive, HH26 is rigidified by a more extensive network of intramolecular salt links and is highly specific, with both association and dissociation rates strongly affected by epitope mutations. HH10 is of intermediate specificity, and epitope mutations produce changes primarily in the dissociation rate. Calorimetric characterization of the association energetics of these three antibodies with the native antigen HEL and with Japanese quail egg white lysozyme (JQL), a naturally occurring avian variant, shows that the energetics of interaction correlate with cross-reactivity and specificity. These results suggest that the greater cross-reactivity of HH8 may be mediated by a combination of conformational flexibility and less specific intermolecular interactions. Thermodynamic calculations suggest that upon association HH8 incurs the largest configurational entropic penalty and also the smallest loss of enthalpic driving force with variant antigen. Much smaller structural perturbations are expected in the formation of the less flexible HH26 complex, and the large loss of enthalpic driving force observed with variant antigen reflects its specificity. The observed thermodynamic parameters correlate well with the observed functional behavior of the antibodies and illustrate fundamental differences in thermodynamic characteristics between cross-reactive and specific molecular recognition.

Conserved distal loop residues in the Hsp104 and ClpB middle domain contact nucleotide-binding domain 2 and enable Hsp70-dependent protein disaggregation

Background: How Hsp104 and ClpB coordinate polypeptide handover with Hsp70 to dissolve disordered protein aggregates is unknown. Results: Conserved distal loop residues in the Hsp104 and ClpB middle domain contact NBD2 and enable Hsp70-dependent protein disaggregation. Conclusion: Distal loop does not project out into solution and Hsp104 and ClpB are tuned differently for Hsp70 collaboration. Significance: Understanding how protein disaggregases operate may empower strategies to counter protein-misfolding disorders. 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.

Purification of Hsp104, a Protein Disaggregase

Hsp104 is a hexameric AAA+ protein(1) from yeast, which couples ATP hydrolysis to protein disaggregation (Fig. 1). This activity imparts two key selective advantages. First, renaturation of disordered aggregates by Hsp104 empowers yeast survival after various protein-misfolding stresses, including heat shock. Second, remodeling of cross-beta amyloid fibrils by Hsp104 enables yeast to exploit myriad prions (infectious amyloids) as a reservoir of beneficial and heritable phenotypic variation. Remarkably, Hsp104 directly remodels preamyloid oligomers and amyloid fibrils, including those comprised of the yeast prion proteins Sup35 and Ure2). This amyloid-remodeling functionality is a specialized facet of yeast Hsp104. The E. coli orthologue, ClpB, fails to remodel preamyloid oligomers or amyloid fibrils. Hsp104 orthologues are found in all kingdoms of life except, perplexingly, animals. Indeed, whether animal cells possess any enzymatic system that couples protein disaggregation to renaturation (rather than degradation) remains unknown. Thus, we and others have proposed that Hsp104 might be developed as a therapeutic agent for various neurodegenerative diseases connected with the misfolding of specific proteins into toxic preamyloid oligomers and amyloid fibrils. There are no treatments that directly target the aggregated species associated with these diseases. Yet, Hsp104 dissolves toxic oligomers and amyloid fibrils composed of alpha-synuclein, which are connected with Parkinsons Disease as well as amyloid forms of PrP. Importantly, Hsp104 reduces protein aggregation and ameliorates neurodegeneration in rodent models of Parkinsons Disease and Huntingtons disease. Ideally, to optimize therapy and minimize side effects, Hsp104 would be engineered and potentiated to selectively remodel specific aggregates central to the disease in question. However, the limited structural and mechanistic understanding of how Hsp104 disaggregates such a diverse repertoire of aggregated structures and unrelated proteins frustrates these endeavors. To understand the structure and mechanism of Hsp104, it is essential to study the pure protein and reconstitute its disaggregase activity with minimal components. Hsp104 is a 102 kDa protein with a pI of -5.3, which hexamerizes in the presence of ADP or ATP, or at high protein concentrations in the absence of nucleotide. Here, we describe an optimized protocol for the purification of highly active, stable Hsp104 from E. coli. The use of E. coli allows simplified large-scale production and our method can be performed quickly and reliably for numerous Hsp104 variants. Our protocol increases Hsp104 purity and simplifies His(6)-tag removal compared to a previous purification method from E. coli. Moreover, our protocol is more facile and convenient than two more recent protocols.

Preventing Parkinson's pathology

Parkinson’s disease (PD) is a progressive neurodegenerative disorder caused by the loss of dopaminergic neurons in the substantia nigra pars compacta of the midbrain. The impaired production and secretion of dopamine causes a variety of symptoms, including bradykinesia, tremor, rigidity, and other

Investigation Of A 6-fluorotryptophan Substituted scFv

For many years our laboratory has pursued an understanding of the protein characteristics which confer specificity and affinity to the antibody for its antigen using a family of monoclonal antibodies to hen egg white lysozyme (HyHEL26, 10, 8 and 63, primarily.) We find that the binding is best characterized by a two-step model representing an association complex becoming a docked complex, evidencing a conformational change.In a recently produced scFv variant of HyHEL10 in which all the tryptophans were substituted with the 6-fluoro form we studied kinetic behavior by Biacore SPR, using our usual protocol to obtain kinetic characterization. We observed that the affinity to lysozyme was concentration dependant, though it did not reflect oligomerization; it changes gradually, allowing investigation, decreasing by an order of magnitude over a period of 3 hours and that most of the change is due to the decrease in the docking step. This repeatable behavior is reversed upon sample reconcentration and delayed by cold. To explore the possible role of folding or water movement we investigated the impact of TMAO, glycerol and some detergents. We also did further exploration by SPR, fluorescence spectroscopy, and other biophysical characterizations in order to better understand the molecular events responsible for this dramatic affinity change.