Dimerization and autophosphorylation of the MST family of kinases are controlled by the same set of residues.

The Hippo pathway controls tissue growth and regulates stem cell fate through the activities of core kinase cassette that begins with the Sterile 20-like kinase MST1/2. Activation of MST1/2 relies on trans-autophosphorylation but the details of the mechanisms regulating that reaction are not fully elucidated. Proposals include dimerization as a first step and include multiple models for potential kinase-domain dimers. Efforts to verify and link these dimers to trans-autophosphorylation were unsuccessful. We explored the link between dimerization and trans-autophosphorylation for MST2 and the entire family of MST kinases. We analyzed crystal lattice contacts of structures of MST kinases and identified an ensemble of kinase-domain dimers compatible with trans-autophosphorylation. These dimers share a common dimerization interface comprised of the activation loop and αG-helix while the arrangements of the kinase-domains within the dimer varied depending on their activation state. We then verified the dimerization interface and determined its function using MST2. Variants bearing alanine substitutions of the αG-helix prevented dimerization of the MST2 kinase domain both in solution and in cells. These substitutions also blocked autophosphorylation of full-length MST2 and its Drosophila homolog Hippo in cells. These variants retain the same secondary structure as wild-type and capacity to phosphorylate a protein substrate, indicating the loss of MST2 activation can be directly attributed to a loss of dimerization rather than loss of either fold or catalytic function. Together this data functionally links dimerization and autophosphorylation for MST2 and suggests this activation mechanism is conserved across both species and the entire MST family.

Consensus sequence design as a general strategy to create hyperstable, biologically active proteins.

Consensus sequence design offers a promising strategy for designing proteins of high stability while retaining biological activity since it draws upon an evolutionary history in which residues important for both stability and function are likely to be conserved. Although there have been several reports of successful consensus design of individual targets, it is unclear from these anecdotal studies how often this approach succeeds and how often it fails. Here, we attempt to assess generality by designing consensus sequences for a set of six protein families with a range of chain lengths, structures, and activities. We characterize the resulting consensus proteins for stability, structure, and biological activities in an unbiased way. We find that all six consensus proteins adopt cooperatively folded structures in solution. Strikingly, four of six of these consensus proteins show increased thermodynamic stability over naturally occurring homologs. Each consensus protein tested for function maintained at least partial biological activity. Although peptide binding affinity by a consensus-designed SH3 is rather low, Km values for consensus enzymes are similar to values from extant homologs. Although consensus enzymes are slower than extant homologs at low temperature, they are faster than some thermophilic enzymes at high temperature. An analysis of sequence properties shows consensus proteins to be enriched in charged residues, and rarified in uncharged polar residues. Sequence differences between consensus and extant homologs are predominantly located at weakly conserved surface residues, highlighting the importance of these residues in the success of the consensus strategy.

Surface residues and nonadditive interactions stabilize a consensus homeodomain protein.

Despite the widely reported success of consensus design in producing highly stabilized proteins, little is known about the physical mechanisms underlying this stabilization. Here, we explore the potential sources of stabilization by performing a systematic analysis of the 29 substitutions that we previously found to collectively stabilize a consensus homeodomain compared with an extant homeodomain. By separately introducing groups of consensus substitutions that alter or preserve charge state, occur at varying degrees of residue burial, and occur at positions of varying degrees of conservation, we determine the extent to which these three features contribute to the consensus stability enhancement. Surprisingly, we find that the largest total contribution to stability comes from consensus substitutions on the protein surface and that the largest per substitution contributions come from substitutions that maintain charge state. This finding suggests that, although consensus proteins are often enriched in charged residues, consensus stabilization does not result primarily from interactions involving charged residues. Although consensus substitutions at strongly conserved positions also contribute disproportionately to stabilization, significant stabilization is also contributed from substitutions at weakly conserved positions. Furthermore, we find that identical consensus substitutions show larger stabilizing effects when introduced into the consensus background than when introduced into an extant homeodomain, indicating that synergistic, stabilizing interactions among the consensus residues contribute to consensus stability enhancement of the homeodomain. By measuring DNA binding affinity for the same set of variants, we find that, although consensus design of the homeodomain increases both affinity and folding stability, it does so using a largely nonoverlapping set of substitutions.

Biophysical characterization of SARAH domain-mediated multimerization of Hippo pathway complexes in Drosophila.

Hippo pathway signaling limits cell growth and proliferation and maintains the stem-cell niche. These cellular events result from the coordinated activity of a core kinase cassette that is regulated, in part, by interactions involving Hippo, Salvador, and dRassF. These interactions are mediated by a conserved coiled-coil domain, termed SARAH, in each of these proteins. SARAH domain-mediated homodimerization of Hippo kinase leads to autophosphorylation and activation. Paradoxically, SARAH domain-mediated heterodimerization between Hippo and Salvador enhances Hippo kinase activity in cells, whereas complex formation with dRassF inhibits it. To better understand the mechanism by which each complex distinctly modulates Hippo kinase and pathway activity, here we biophysically characterized the entire suite of SARAH domain-mediated complexes. We purified the three SARAH domains from Drosophila melanogaster and performed an unbiased pulldown assay to identify all possible interactions, revealing that isolated SARAH domains are sufficient to recapitulate the cellular assemblies and that Hippo is a universal binding partner. Additionally, we found that the Salvador SARAH domain homodimerizes and demonstrate that this interaction is conserved in Salvador's mammalian homolog. Using native MS, we show that each of these complexes is dimeric in solution. We also measured the stability of each SARAH domain complex, finding that despite similarities at both the sequence and structural levels, SARAH domain complexes differ in stability. The identity, stoichiometry, and stability of these interactions characterized here comprehensively reveal the nature of SARAH domain-mediated complex formation and provide mechanistic insights into how SARAH domain-mediated interactions influence Hippo pathway activity.

Creating a Homeodomain with High Stability and DNA Binding Affinity by Sequence Averaging.

There is considerable interest in generating proteins with both high stability and high activity for biomedical and industrial purposes. One approach that has been used successfully to increase the stability of linear repeat proteins is consensus design. It is unclear the extent over which the consensus design approach can be used to produce folded and hyperstable proteins, and importantly, whether such stabilized proteins would retain function. Here we extend the consensus strategy to design a globular protein. We show that a consensus-designed homeodomain (HD) sequence adopts a cooperatively folded homeodomain structure. The unfolding free energy of the consensus-HD is 5 kcal·mol-1 higher than that of the naturally occurring engrailed-HD from Drosophila melanogaster. Remarkably, the consensus-HD binds the engrailed-HD cognate DNA in a similar mode as the engrailed-HD with approximately 100-fold higher affinity. 15N relaxation studies show a decrease in ps-ns backbone dynamics in the free state of consensus-HD, suggesting that increased affinity is not a result of increased plasticity. In addition to demonstrating the potential for consensus design of globular proteins with increased stability, these results demonstrate that greatly stabilized proteins can bind cognate substrates with increased affinities, showing that high stability is compatible with function.

Dimerization and autophosphorylation of the MST family of kinases are controlled by the same set of residues.

The Hippo pathway controls tissue growth and regulates stem cell fate through the activities of core kinase cassette that begins with the Sterile 20-like kinase MST1/2. Activation of MST1/2 relies on trans -autophosphorylation but the details of the mechanisms regulating that reaction are not fully elucidated. Proposals include dimerization as a first step and include multiple models for potential kinase-domain dimers. Efforts to verify and link these dimers to trans -autophosphorylation were unsuccessful. We explored the link between dimerization and trans -autophosphorylation for MST2 and the entire family of MST kinases. We analyzed crystal lattice contacts of structures of MST kinases and identified an ensemble of kinase-domain dimers compatible with trans -autophosphorylation. These dimers share a common dimerization interface comprised of the activation loop and αG-helix while the arrangements of the kinase-domains within the dimer varied depending on their activation state. We then verified the dimerization interface and determined its function using MST2. Variants bearing alanine substitutions of the αG-helix prevented dimerization of the MST2 kinase domain both in solution and in cells. These substitutions also blocked autophosphorylation of full-length MST2 and its Drosophila homolog Hippo in cells. These variants retain the same secondary structure as wild-type and capacity to phosphorylate a protein substrate, indicating the loss of MST2 activation can be directly attributed to a loss of dimerization rather than loss of either fold or catalytic function. Together this data functionally links dimerization and autophosphorylation for MST2 and suggests this activation mechanism is conserved across both species and the entire MST family.

The use of consensus sequence information to engineer stability and activity in proteins.

The goal of protein design is to create proteins that are stable, soluble, and active. Here we focus on one approach to protein design in which sequence information is used to create a "consensus" sequence. Such consensus sequences comprise the most common residue at each position in a multiple sequence alignment (MSA). After describing some general ideas that relate MSA and consensus sequences and presenting a statistical thermodynamic framework that relates consensus and non-consensus sequences to stability, we detail the process of designing a consensus sequence and survey reports of consensus design and characterization from the literature. Many of these consensus proteins retain native biological activities including ligand binding and enzyme activity. Remarkably, in most cases the consensus protein shows significantly higher stability than extant versions of the protein, as measured by thermal or chemical denaturation, consistent with the statistical thermodynamic model. To understand this stability increase, we compare various features of consensus sequences with the extant MSA sequences from which they were derived. Consensus sequences show enrichment in charged residues (most notably glutamate and lysine) and depletion of uncharged polar residues (glutamine, serine, and asparagine). Surprisingly, a survey of stability changes resulting from point substitutions show little correlation with residue frequencies at the corresponding positions within the MSA, suggesting that the high stability of consensus proteins may result from interactions among residue pairs or higher-order clusters. Whatever the source, the large number of reported successes demonstrates that consensus design is a viable route to generating active and in many cases highly stabilized proteins.

Rerouting the folding pathway of the Notch ankyrin domain by reshaping the energy landscape.

The modular nature of repeat proteins has made them a successful target for protein design. Ankyrin repeat, TPR, and leucine rich repeat domains that have been designed solely on consensus information have been shown to have higher thermostability than their biological counterparts. We have previously shown that we can reshape the energy landscape of a repeat protein by adding multiple C-terminal consensus ankyrin repeats to the five N-terminal repeats of the Notch ankyrin domain. Here we explore how the folding mechanism responds to reshaping of the energy landscape. We have used analogous substitutions of a conserved alanine with glycine in each repeat to determine the distribution of structure in the transition state ensembles of constructs containing one (Nank1-5C1) and two consensus (Nank1-5C2) ankyrin repeats. Whereas folding of the wild-type Notch ankyrin domain is slowed by substitutions in its central repeats, (1) folding of Nank1-5C1 and Nank1-5C2 is slowed by substitutions in the C-terminal repeats. Thus, the addition of C-terminal stabilizing repeats shifts the transition state ensemble toward the C-terminal repeats, rerouting the folding pathway of the ankyrin repeat domain. These findings indicate that, for the Notch ankyrin domain, folding pathways are selected based on local energetics.

Enhancing the stability and folding rate of a repeat protein through the addition of consensus repeats.

Repeat proteins are constructed from a linear array of modular units, giving rise to an overall topology lacking long-range interactions. This suggests that stabilizing repeat modules based on consensus information might be added to a repeat protein domain, allowing it to be extended without altering its overall topology. Here we add consensus modules the ankyrin repeat domain from the Drosophila Notch receptor to investigate the structural tolerance to these modules, the relative thermodynamic stability of these hybrid proteins, and how alterations in the energy landscape influence folding kinetics. Insertions of consensus modules between repeats five and six of the Notch ankyrin domain have little effect on the far and near-UV CD spectra, indicating that neither secondary nor tertiary structure is dramatically altered. Furthermore, stable structure is maintained at increased denaturant concentrations in the polypeptides containing the consensus repeats, indicating that the consensus modules are capable of stabilizing much of the domain. However, insertion of the consensus repeats appears to disrupt cooperativity, producing a two-stage (three-state) unfolding transition in which the C-terminal repeats unfold at moderate urea concentrations. Removing the C-terminal repeats (Notch ankyrin repeats six and seven) restores equilibrium two-state folding and demonstrates that the high stability of the consensus repeats is propagated into the N-terminal, naturally occurring Notch ankyrin repeats. This stability increase greatly increases the folding rate, and suggests that the transition state ensemble may be repositioned in the chimeric consensus-stabilized proteins in response to local stability.

Experimental characterization of the folding kinetics of the notch ankyrin domain.

Proteins constructed from linear arrays of tandem repeats provide a simplified architecture for understanding protein folding. Here, we examine the folding kinetics of the ankyrin repeat domain from the Drosophila Notch receptor, which consists of six folded ankyrin modules and a seventh partly disordered N-terminal ankyrin repeat sequence. Both the refolding and unfolding kinetics are best described as a sum of two exponential phases. The slow, minor refolding phase is limited by prolyl isomerization in the denatured state (D). The minor unfolding phase, which appears as a lag during fluorescence-detected unfolding, is consistent with an on-pathway intermediate (I). This intermediate, although not directly detected during refolding, is shown to be populated by interrupted refolding experiments. When plotted against urea, the rate constants for the major unfolding and refolding phases define a single non-linear v-shaped chevron, as does the minor unfolding phase. These two chevrons, along with unfolding amplitudes, are well-fitted by a sequential three-state model, which yields rate constants for the individual steps in folding and unfolding. Based on these fitted parameters, the D to I step is rate-limiting, and closely matches the major observed refolding phase at low denaturant concentrations. I appears to be midway between N and D in folding free energy and denaturant sensitivity, but has Trp fluorescence properties close to N. Although the Notch ankyrin domain has a simple architecture, folding is slow, with the limiting refolding rate constant as much as seven orders of magnitude smaller than expected from topological predictions.

The tolerance of a modular protein to duplication and deletion of internal repeats.

Ankyrin repeat polypeptides contain repeated structural elements that pack to produce modular architectures lacking in close contacts between distant segments of the polypeptide chain. Despite this lack of sequence-distant contacts, ankyrin repeat polypeptides have been shown to fold in a cooperative manner. To determine the distance over which cooperative interactions can be propagated in a repeat protein, and to investigate the tolerance to internal duplication and deletion of modules, we have constructed a series of ankyrin repeat variants of the Notch ankyrin domain in which repeat number is varied by duplication and deletion of internal repeats. A construct with two copies of the fifth ankyrin repeat shows a modest increase in stability compared to the parent construct and retains apparent two-state unfolding behavior. Although constructs containing three and four copies of the fifth repeat retain this increased resistance to urea, they exhibit broad, multi-state unfolding transitions compared to the parent construct. For the Notch ankyrin domain, these larger constructs may represent a limit beyond which full cooperativity cannot be maintained. Deletions of internal repeats from the Notch ankyrin domain significantly destabilize the domain. This severe destabilization, which is larger than that resulting from end-repeat deletion, may arise from unfavorable interactions within the new non-native interfaces produced by internal repeat deletion. These results demonstrate both an asymmetry between the duplication and deletion of internal repeats, and a difference between deletion of internal and end-repeats, suggesting preferred mechanisms for evolution of repeat proteins.

When the Scaffold Cannot Be Ignored: The Role of the Hydrophobic Core in Ligand Binding and Specificity.

The traditional view of protein-ligand binding treats a protein as comprising distinct binding epitopes on the surface of a degenerate structural scaffold, largely ignoring the impact of a protein's energy landscape. To determine the robustness of this simplification, we compared two small helix-turn-helix transcription factors with different energy landscapes. λ-Repressor is stable and well folded, while MarA appears to be marginally stable with multiple native conformations (molten). While λ-repressor is known to tolerate any hydrophobic mutation in the core, we find MarA drastically less tolerant to core mutation. Moreover, core mutations in MarA (distant from the DNA-binding interface) change the relative affinities of its binding partners, altering ligand specificity. These results can be explained by taking into account the effects of mutations on the entire energy landscape and not just the native state. Thus, for proteins with multiple conformations that are close in energy, such as many intrinsically disordered proteins, residues distant from the active site can alter both binding affinity and specificity.

Mono and dual cofactor dependence of human cystathionine ß-synthase enzyme variants in vivo and in vitro.

Any two individuals differ from each other by an average of 3 million single-nucleotide polymorphisms. Some polymorphisms have a functional impact on cofactor-using enzymes and therefore represent points of possible therapeutic intervention through elevated-cofactor remediation. Because most known disease-causing mutations affect protein stability, we evaluated how the in vivo impact caused by single amino acid substitutions in a prototypical enzyme of this type compared with physical characteristics of the variant enzymes in vitro. We focused on cystathionine ß-synthase (CBS) because of its clinical relevance in homocysteine metabolism and because some variants of the enzyme are clinically responsive to increased levels of its B6 cofactor. Single amino-acid substitutions throughout the CBS protein caused reduced function in vivo, and a subset of these altered sensitivity to limiting B6-cofactor. Some of these B6-sensitive substitutions also had altered sensitivity to limiting heme, another CBS cofactor. Limiting heme resulted in reduced incorporation of heme into these variants, and subsequently increased protease sensitivity of the enzyme in vitro. We hypothesize that these alleles caused a modest, yet significant, destabilization of the native state of the protein, and that the functional impact of the amino acid substitutions caused by these alleles can be influenced by cofactor(s) even when the affected amino acid is distant from the cofactor binding site.