DnaK: Structural Functions and Mutant Protein Quality Control

Proteins must fold into precise three-dimensional shapes to function correctly, but many face challenges in achieving or maintaining this structure. Molecular chaperones assist in protein folding and prevent misfolding-related damage, with DnaK playing a critical role in bacterial systems. This heat shock protein is essential for managing cellular stress and ensuring proteins reach their functional form.

Understanding how DnaK interacts with mutant proteins provides insight into its role in quality control and proteostasis. Researchers examine its structural properties, mechanisms of action, and interactions with co-chaperones to explore potential applications in biotechnology and medicine.

Structural And Functional Properties

DnaK, part of the Hsp70 family, is an ATP-dependent molecular chaperone that facilitates protein folding and stabilization in bacterial cells. It consists of two primary domains: the N-terminal nucleotide-binding domain (NBD) and the C-terminal substrate-binding domain (SBD). The NBD hydrolyzes ATP to ADP, driving conformational changes that regulate substrate affinity, while the SBD binds exposed hydrophobic regions of unfolded proteins. This ATP-driven cycle allows DnaK to transition between high- and low-affinity states, ensuring efficient protein folding.

DnaK undergoes significant conformational shifts during its chaperone cycle. In its ATP-bound state, the SBD adopts an open conformation, allowing substrate exchange. ATP hydrolysis triggers a closed state that secures the polypeptide, providing a protected environment for folding. Structural studies using X-ray crystallography and cryo-electron microscopy have detailed these conformational changes, emphasizing interdomain flexibility in DnaK’s function.

DnaK’s substrate-binding domain is highly adaptable, enabling it to recognize a diverse range of polypeptides. Unlike specific enzymes, DnaK interacts with a broad spectrum of unfolded or partially folded proteins, targeting hydrophobic residues usually buried in native structures. This selective recognition prevents aggregation and facilitates proper folding, particularly under stress conditions. The SBD’s β-sheet architecture and flexible lid domain enhance its ability to accommodate substrates of varying sizes and sequences, reinforcing its role as a generalist chaperone.

Mechanism Of Action With Mutant Proteins

DnaK stabilizes misfolded mutant proteins that expose hydrophobic residues typically buried in their native state, preventing aggregation. This process is driven by ATP-dependent cycling, where DnaK alternates between high- and low-affinity states to facilitate substrate binding and release. The efficiency of this mechanism varies with the mutation, with some variants requiring prolonged chaperone interaction to achieve proper folding.

Destabilizing mutations alter folding kinetics, often trapping proteins in intermediate states prone to misfolding or degradation. DnaK provides a transiently stable environment, allowing proteins to attempt refolding. Studies on temperature-sensitive mutants in bacterial enzymes show that DnaK can restore function by stabilizing active conformations at non-permissive temperatures.

DnaK also plays a role in protein degradation when refolding fails. If a protein remains misfolded despite multiple attempts, DnaK recruits degradation machinery to target it for proteolysis. This triage function is particularly relevant for highly unstable or aggregation-prone mutants. Studies on cystic fibrosis transmembrane conductance regulator (CFTR) mutants and bacterial transcription factors suggest that DnaK influences whether a mutant protein is salvaged or degraded, with outcomes depending on mutation severity, cellular stress, and co-chaperone availability.

Role In Intracellular Quality Control

DnaK functions as a surveillance system, monitoring intracellular proteins for misfolding. By recognizing exposed hydrophobic regions, it differentiates between properly folded and misfolded proteins, ensuring only functional structures persist. This role is especially critical under thermal or chemical stress, where misfolded proteins accumulate.

Beyond preventing aggregation, DnaK determines the fate of aberrant proteins. Some can be refolded through multiple chaperone-assisted cycles, while others persist in misfolded states. In such cases, DnaK works with proteolytic systems to direct irreversibly damaged proteins toward degradation. The ClpB-DnaK partnership is particularly important, as ClpB disaggregates protein clusters, allowing DnaK to either refold salvageable proteins or guide them toward protease recognition.

DnaK’s efficiency in maintaining protein quality control depends on ATP availability, co-chaperone interactions, and cellular stress levels. Under normal conditions, its activity is tightly regulated. However, during heat shock or oxidative stress, DnaK expression increases to manage a higher load of misfolded substrates. Escherichia coli models show that mutations disrupting DnaK impair proteostasis, underscoring its essential role in intracellular quality control.

Interactions With Co-Chaperone Systems

DnaK operates within a network of co-chaperones that regulate its activity. The most prominent are DnaJ and GrpE, which influence its ATPase cycle and substrate binding. DnaJ, an Hsp40 family member, recognizes misfolded polypeptides and delivers them to DnaK. By binding exposed hydrophobic regions, DnaJ stimulates DnaK’s ATP hydrolysis, promoting a high-affinity substrate-bound state.

GrpE acts as a nucleotide exchange factor, accelerating ADP release and resetting DnaK to its ATP-bound state. This step is crucial for enabling multiple rounds of chaperone-assisted folding. The interplay between DnaJ, DnaK, and GrpE fine-tunes substrate stabilization and turnover, optimizing folding efficiency. Escherichia coli studies indicate that disruptions in this co-chaperone system significantly reduce DnaK’s chaperoning capacity.

Techniques For Studying DnaK With Protein Mutants

Investigating DnaK’s interaction with mutant proteins requires biochemical, structural, and genetic approaches. Researchers use diverse experimental techniques to examine how DnaK recognizes, binds, and stabilizes protein variants.

Structural analyses, including X-ray crystallography and cryo-electron microscopy, have mapped the ATP-dependent shifts between open and closed states, detailing how mutant substrates interact with the substrate-binding domain. Nuclear magnetic resonance (NMR) spectroscopy captures transient interactions in solution, providing insights into how destabilized proteins engage with DnaK in real time.

Biochemical assays such as fluorescence anisotropy and surface plasmon resonance quantify DnaK’s binding kinetics with mutant proteins, revealing how specific mutations influence chaperone-substrate stability. In vivo methods, including bacterial growth assays and proteomic analyses, assess the functional impact of DnaK interactions within cells. By combining these techniques, researchers gain a comprehensive understanding of how DnaK mitigates folding defects caused by genetic mutations.