Identifying Site U: An Uncharacterized DnaK Binding Region in proPhoA

Presenter: Emilio Salazar

Faculty Sponsor: Lila M. Gierasch

School: UMass Amherst

Research Area: Biochemistry and Molecular Biology

Session: Poster Session 5, 3:15 PM - 4:00 PM, Concourse, B5

ABSTRACT

Hsp70 molecular chaperones are crucial for maintaining cellular protein health. The substrate selectivity of Hsp70s is notable due to their paradoxical ability to bind a multitude of clients while discerning between folded and unfolded proteins. Clerico et al., 2021, investigated this puzzle through binding studies of an unfolded substrate protein, alkaline phosphatase precursor (proPhoA), to the substrate-binding domain (SBD) of the E. coli Hsp70, DnaK. NMR data confirmed the proPhoA binding sites previously discovered through peptide arrays, but also revealed a novel DnaK binding site, Site U. Notably, chemical shifts observed for the δ1-methyls of isoleucines 401 and 438, which reside in the substrate-binding cleft of the SBD, correlate with presence of a leucine residue in the center of bound U. In addition, titration of proPhoA with SBD reveals that U has a relatively high affinity interaction with DnaK. I hypothesize that DnaK’s affinity for Site U is dependent on avidity, which is lost in the short motifs previously employed. To narrow down the region of proPhoA that contains Site U, NMR was again performed on labeled SBD, with a total of six different proPhoA fragments that are long enough to allow for multivalent interactions with DnaK. Once the region containing site U is identified, this will be followed up by crosslinking mass spectrometry. This technique will narrow the location of Site U from an entire fragment to a few residues. The results will shed light on how an Hsp70 molecular chaperone selects its binding sites.

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