Daniel N. Bolon

Sculpting the Proteome with AAA+ Proteases and Disassembly Machines

Machines of protein destruction-including energy-dependent proteases and disassembly chaperones of the AAA(+) ATPase family-function in all kingdoms of life to sculpt the cellular proteome, ensuring that unnecessary and dangerous proteins are eliminated and biological responses to environmental change are rapidly and properly regulated. Exciting progress has been made in understanding how AAA(+) machines recognize specific proteins as targets and then carry out ATP-dependent dismantling of the tertiary and/or quaternary structure of these molecules during the processes of protein degradation and the disassembly of macromolecular complexes.

Enzyme-like proteins by computational design

We report the development and initial experimental validation of a computational design procedure aimed at generating enzyme-like protein catalysts called “protozymes.” Our design approach utilizes a “compute and build” strategy that is based on the physical/chemical principles governing protein stability and catalytic mechanism. By using the catalytically inert 108-residue Escherichia coli thioredoxin as a scaffold, the histidine-mediated nucleophilic hydrolysis of p-nitrophenyl acetate as a model reaction, and the ORBIT protein design software to compute sequences, an active site scan identified two promising catalytic positions and surrounding active-site mutations required for substrate binding. Experimentally, both candidate protozymes demonstrated catalytic activity significantly above background. One of the proteins, PZD2, displayed “burst” phase kinetics at high substrate concentrations, consistent with the formation of a stable enzyme intermediate. The kinetic parameters of PZD2 are comparable to early catalytic Abs. But, unlike catalytic Ab design, our design procedure is independent of fold, suggesting a possible mechanism for examining the relationships between protein fold and the evolvability of protein function.

Experimental illumination of a fitness landscape

The genes of all organisms have been shaped by selective pressures. The relationship between gene sequence and fitness has tremendous implications for understanding both evolutionary processes and functional constraints on the encoded proteins. Here, we have exploited deep sequencing technology to experimentally determine the fitness of all possible individual point mutants under controlled conditions for a nine-amino acid region of Hsp90. Over the past five decades, limited glimpses into the relationship between gene sequence and function have sparked a long debate regarding the distribution, relative proportion, and evolutionary significance of deleterious, neutral, and advantageous mutations. Our systematic experimental measurement of fitness effects of Hsp90 mutants in yeast, evaluated in the light of existing population genetic theory, are remarkably consistent with a nearly neutral model of molecular evolution.

Asymmetric interactions of ATP with the AAA+ Clpx6 unfoldase : Allosteric control of a protein machine

ATP hydrolysis by AAA+ ClpX hexamers powers protein unfolding and translocation during ClpXP degradation. Although ClpX is a homohexamer, positive and negative allosteric interactions partition six potential nucleotide binding sites into three classes with asymmetric properties. Some sites release ATP rapidly, others release ATP slowly, and at least two sites remain nucleotide free. Recognition of the degradation tag of protein substrates requires ATP binding to one set of sites and ATP or ADP binding to a second set of sites, suggesting a mechanism that allows repeated unfolding attempts without substrate release over multiple ATPase cycles. Our results rule out concerted hydrolysis models involving ClpX(6)*ATP(6) or ClpX(6)*ADP(6) and highlight structures of hexameric AAA+ machines with three or four nucleotides as likely functional states. These studies further emphasize commonalities between distant AAA+ family members, including protein and DNA translocases, helicases, motor proteins, clamp loaders, and other ATP-dependent enzymes.

De novo design of biocatalysts

The challenging field of de novo enzyme design is beginning to produce exciting results. The application of powerful computational methods to functional protein design has recently succeeded at engineering target activities. In addition, efforts in directed evolution continue to expand the transformations that can be accomplished by existing enzymes. The engineering of completely novel catalytic activity requires traversing inactive sequence space in a fitness landscape, a feat that is better suited to computational design. Optimizing activity, which can include subtle alterations in backbone conformation and protein motion, is better suited to directed evolution, which is highly effective at scaling fitness landscapes towards maxima. Improved rational design efforts coupled with directed evolution should dramatically improve the scope of de novo enzyme design.

Analyses of the effects of all ubiquitin point mutants on yeast growth rate

The amino acid sequence of a protein governs its function. We used bulk competition and focused deep sequencing to investigate the effects of all ubiquitin point mutants on yeast growth rate. Many aspects of ubiquitin function have been carefully studied, which enabled interpretation of our growth analyses in light of a rich structural, biophysical and biochemical knowledge base. In one highly sensitive cluster on the surface of ubiquitin, almost every amino acid substitution caused growth defects. In contrast, the opposite face tolerated virtually all possible substitutions. Surface locations between these two faces exhibited intermediate mutational tolerance. The sensitive face corresponds to the known interface for many binding partners. Across all surface positions, we observe a strong correlation between burial at structurally characterized interfaces and the number of amino acid substitutions compatible with robust growth. This result indicates that binding is a dominant determinant of ubiquitin function. In the solvent-inaccessible core of ubiquitin, all positions tolerated a limited number of substitutions, with hydrophobic amino acids especially interchangeable. Some mutations null for yeast growth were previously shown to populate folded conformations indicating that, for these mutants, subtle changes to conformation caused functional defects. The most sensitive region to mutation within the core was located near the C-terminus that is a focal binding site for many critical binding partners. These results indicate that core mutations may frequently cause functional defects through subtle disturbances to structure or dynamics.

Flexible Linkers Leash the Substrate Binding Domain of SspB to a Peptide Module that Stabilizes Delivery Complexes with the AAA+ ClpXP Protease

SspB dimers bind proteins bearing the ssrA-degradation tag and stimulate their degradation by the ClpXP protease. Here, E. coli SspB is shown to contain a dimeric substrate binding domain of 110-120 N-terminal residues, which binds ssrA-tagged substrates but does not stimulate their degradation. The C-terminal 40-50 residues of SspB are unstructured but are required for SspB to form substrate-delivery complexes with ClpXP. A synthetic peptide containing the 10 C-terminal residues of SspB binds ClpX, stimulates its ATPase activity, and prevents SspB-mediated delivery of GFP-ssrA for ClpXP degradation. This tripartite structure--an ssrA-tag binding and dimerization domain, a flexible linker, and a short peptide module that docks with ClpX--allows SspB to deliver tagged substrates to ClpXP without interfering with their denaturation or degradation.

Bivalent Tethering of SspB to ClpXP Is Required for Efficient Substrate Delivery: A Protein-Design Study

SspB homodimers deliver ssrA-tagged substrates to ClpXP for degradation. SspB consists of a substrate binding domain and an unstructured tail with a ClpX binding module (XB). Using computational design, we engineered an SspB heterodimer whose subunits did not form homodimers. Experiments with the designed molecule and variants lacking one or two tails demonstrate that both XB modules are required for strong binding and efficient substrate delivery to ClpXP. Assembly of stable SspB-substrate-ClpX delivery complexes requires the coupling of weak tethering interactions between ClpX and the SspB XB modules as well as interactions between ClpX and the substrate degradation tag. The ClpX hexamer contains three XB binding sites, one per N domain dimer, and thus binds strongly to just one SspB dimer at a time. Because different adaptor proteins use the same tethering sites in ClpX, those which employ bivalent tethering, like SspB, will compete more effectively for substrate delivery to ClpXP.

Influenza Virus Drug Resistance: A Time-Sampled Population Genetics Perspective

The challenge of distinguishing genetic drift from selection remains a central focus of population genetics. Time-sampled data may provide a powerful tool for distinguishing these processes, and we here propose approximate Bayesian, maximum likelihood, and analytical methods for the inference of demography and selection from time course data. Utilizing these novel statistical and computational tools, we evaluate whole-genome datasets of an influenza A H1N1 strain in the presence and absence of oseltamivir (an inhibitor of neuraminidase) collected at thirteen time points. Results reveal a striking consistency amongst the three estimation procedures developed, showing strongly increased selection pressure in the presence of drug treatment. Importantly, these approaches re-identify the known oseltamivir resistance site, successfully validating the approaches used. Enticingly, a number of previously unknown variants have also been identified as being positively selected. Results are interpreted in the light of Fishers Geometric Model, allowing for a quantification of the increased distance to optimum exerted by the presence of drug, and theoretical predictions regarding the distribution of beneficial fitness effects of contending mutations are empirically tested. Further, given the fit to expectations of the Geometric Model, results suggest the ability to predict certain aspects of viral evolution in response to changing host environments and novel selective pressures.

Shifting fitness landscapes in response to altered environments.

The role of adaptation in molecular evolution has been contentious for decades. Here, we shed light on the adaptive potential in Saccharomyces cerevisiae by presenting systematic fitness measurements for all possible point mutations in a region of Hsp90 under four environmental conditions. Under elevated salinity, we observe numerous beneficial mutations with growth advantages up to 7% relative to the wild type. All of these beneficial mutations were observed to be associated with high costs of adaptation. We thus demonstrate that an essential protein can harbor adaptive potential upon an environmental challenge, and report a remarkable fit of the data to a version of Fishers geometric model that focuses on the fitness trade‐offs between mutations in different environments.