Biology subjects

Fenton, A. W.

Publications and source records attributed to Fenton, A. W..

8 recordsLinked to original sources

bioRxiv2023

Fructose-1-kinase has pleiotropic roles in Escherichia coli

In Escherichia coli, the master transcription regulator Catabolite Repressor Activator (Cra) regulates >100 genes in central metabolism. Cra binding to DNA is allosterically regulated by binding to fructose-1-phosphate (F-1-P), but the only documented source of F-1-P is from the concurrent import and phosphorylation of exogenous fructose. Thus, many have proposed that fructose-1,6-bisphosphate (F-1,6-BP) is also a physiological regulatory ligand. However, the role of F-1,6-BP has been widely debated. Here, we report that the E. coli enzyme fructose-1-kinase (FruK) can carry out its "reverse" reaction under physiological substrate concentrations to generate F-1-P from F-1,6-BP. We further show that FruK directly binds Cra with nanomolar affinity and forms higher order, heterocomplexes. Growth assays with a {Delta}fruK strain and fruK complementation show that FruK has a broader role in metabolism than fructose catabolism. The {Delta}fruK strain also alters biofilm formation. Since fruK itself is repressed by Cra, these newly-reported events add layers to the dynamic regulation of E. coli central metabolism that occur in response to changing nutrients. These findings might have wide-spread relevance to other {gamma}-proteobacteria, which conserve both Cra and FruK.

microbiology↗

bioRxiv2023

The intrinsically disordered transcriptional activation domain of CIITA is functionally tuneable by single substitutions: An exception or a new rule?

During protein evolution, some amino acid substitutions modulate protein function ("tuneability"). In most proteins, the tuneable range is wide and can be sampled by a set of protein variants that each contains multiple amino acid substitutions. In other proteins, the full tuneable range can be accessed by a set of variants that each contains a single substitution. Indeed, in some globular proteins, the full tuneable range can be accessed by the set of site-saturating substitutions at an individual "rheostat" position. However, in proteins with intrinsically disordered regions (IDRs), most functional studies - which would also detect tuneability - used multiple substitutions or small deletions. In disordered transcriptional activation domains (ADs), studies with multiple substitutions led to the "acidic exposure" model, which does not anticipate the existence of rheostat positions. In the few studies that did assess effects of single substitutions on AD function, results were mixed: The ADs of two full-length transcription factors did not show tuneability, whereas a fragment of a third AD was tuneable by single substitutions. Here, we tested tuneability in the AD of full-length human class II transactivator (CIITA). Sequence analyses and experiments showed that CIITAs AD is an IDR. Functional assays of singly-substituted AD variants showed that CIITAs function was highly tuneable, with outcomes not predicted by the acidic exposure model. Four tested positions showed rheostat behaviour for transcriptional activation. Thus, tuneability of different IDRs can vary widely. Future studies are needed to illuminate the biophysical features that govern whether an IDR is tuneable by single substitutions.

biochemistry↗

bioRxiv2020

"Multiplex" rheostat positions cluster around allosterically critical regions of the lactose repressor protein

Amino acid variation at "rheostat" positions provides opportunity to modulate various aspects of protein function - such as binding affinity or allosteric coupling - across a wide range. Previously a subclass of "multiplex" rheostat positions was identified at which substitutions simultaneously modulated more than one functional parameter. Using the Miller laboratorys dataset of [~]4000 variants of lactose repressor protein (LacI), we compared the structural properties of multiplex rheostat positions with (i) "single" rheostat positions that modulate only one functional parameter, (ii) "toggle" positions that follow textbook substitution rules, and (iii) "neutral" positions that tolerate any substitution without changing function. The combined rheostat classes comprised >40% of LacI positions, more than either toggle or neutral positions. Single rheostat positions were broadly distributed over the structure. Multiplex rheostat positions structurally overlapped with positions involved in allosteric regulation. When their phenotypic outcomes were interpreted within a thermodynamic framework, functional changes at multiplex positions were uncorrelated. This suggests that substitutions lead to complex changes in the underlying molecular biophysics. Bivariable and multivariable analyses of evolutionary signals within multiple sequence alignments could not differentiate single and multiplex rheostat positions. Phylogenetic analyses - such as ConSurf - could distinguish rheostats from toggle and neutral positions. Multivariable analyses could also identify a subset of neutral positions with high probability. Taken together, these results suggest that detailed understanding of the underlying molecular biophysics, likely including protein dynamics, will be required to discriminate single and multiplex rheostat positions from each other and to predict substitution outcomes at these sites.

biochemistry↗

bioRxiv2020

A clinically-relevant polymorphism in the Na+/taurocholate cotransporting polypeptide (NTCP) occurs at a rheostat position

Conventionally, most amino acid substitutions at important protein positions are expected to abolish function. However, in several soluble-globular proteins, we identified a class of non-conserved positions for which various substitutions produced progressive functional changes; we consider these evolutionary “rheostats”. Here, we report a strong rheostat position in the integral membrane protein, Na+/taurocholate cotransporting polypeptide (NTCP), at the site of a pharmacologically-relevant polymorphism (S267F). Functional studies were performed for all 20 substitutions (“S267X”) with three substrates (taurocholate, estrone-3-sulfate and rosuvastatin). The S267X set showed strong rheostatic effects on overall transport, and individual substitutions showed varied effects on transport kinetics (Km and Vmax). However, the outcomes were substrate dependent, indicating altered specificity. To assess protein stability, we measured surface expression and used the Rosetta software suite to model structure and stability changes of S267X. Although buried near the substrate binding site, S267X substitutions were easily accommodated in the NTCP structure model. Across the modest range of changes, calculated stabilities correlated with surface-expression differences, but neither parameter correlated with altered transport. Thus, substitutions at rheostat position 267 had wide-ranging effects on the phenotype of this integral membrane protein. We further propose that polymorphic positions in other proteins might be locations of rheostat positions.Competing Interest StatementThe authors have declared no competing interest.View Full Text

biochemistry↗

bioRxiv2019

Mutational mimics of allosteric effectors: a genome editing design to validate allosteric drug targets

Development of drugs that allosterically regulate enzyme functions to treat disease is a costly venture. Screening mutations that mimic allosteric effectors in vitro will identify therapeutic regulatory targets enhancing the likelihood of developing a disease treatment at a reasonable cost. We demonstrate the potential of this approach utilizing human liver pyruvate kinase (hLPYK) as a model. Inhibition of hLPYK was the first desired outcome of our screen. We identified individual point mutations that: 1) mimicked allosteric inhibition by alanine, 2) mimicked inhibition by protein phosphorylation, and 3) prevented binding of fructose-1,6-bisphosphate (Fru-1,6-BP). Our second desired screening outcome was activation of hLPYK. We identified individual point mutations that: 1) prevented hLPYK from binding alanine, the allosteric inhibitor, 2) prevented inhibitory protein phosphorylation, or 3) mimicked allosteric activation by Fru-1,6-BP. Combining the three activating point mutations produced a constitutively activated enzyme that was unresponsive to regulators. Expression of a mutant hLPYK transgene containing these three mutations in a mouse model was not lethal. Thus, mutational mimics of allosteric effectors will be useful to confirm whether allosteric activation of hLPYK will control glycolytic flux in the diabetic liver to reduce hepatic glucose production and, in turn, reduce or prevent hyperglycemia.

biochemistry↗

bioRxiv2019

Functional tunability from a distance: Rheostat positions influence allosteric coupling between two distant binding sites

For protein mutagenesis, a common expectation is that important positions will behave like on/off \"toggle\" switches (i.e., a few substitutions act like wildtype, most abolish function). However, there exists another class of important positions that manifests a wide range of functional outcomes upon substitution: \"rheostat\" positions. Previously, we evaluated rheostat positions located near the allosteric binding sites for inhibitor alanine (Ala) and activator fructose-1,6-bisphosphate (Fru-1,6-BP) in human liver pyruvate kinase. When substituted with multiple amino acids, many positions demonstrated moderate rheostat effects on allosteric coupling between effector binding and phosphoenolpyruvate (PEP) binding in the active site. Nonetheless, the combined outcomes of all positions sampled the full range of possible allosteric coupling (full tunability). However, that study only evaluated allosteric tunability of \"local\" positions, i.e., positions were located near the binding sites of the allosteric ligand being assessed. Here, we evaluated tunability of allosteric coupling when mutated sites were distant from the allosterically-coupled binding sites. Positions near the Ala binding site had rheostat outcomes on allosteric coupling between Fru-1,6-BP and PEP binding. In contrast, positions in the Fru-1,6-BP site exhibited modest effects on coupling between Ala and PEP binding. Analyzed in aggregate, both PEP/Ala and PEP/Fru-1,6-BP coupling were again fully tunable by amino acid substitutions at this limited set of distant positions. Furthermore, some positions exhibited rheostatic control over multiple parameters and others exhibited rheostatic effects on one parameter and toggle control over a second. These findings highlight challenges in efforts to both predict/interpret mutational outcomes and engineer functions into proteins.

biochemistry↗

bioRxiv2019

Do neutral protein positions really exist? A case study with allostery in human liver pyruvate kinase

Understanding how each residue position contributes to protein function has been a long-standing goal in protein science. Substitution studies have historically focused on conserved protein positions. However, substitutions of nonconserved positions can also modify function. Indeed, we recently identified nonconserved positions that have large substitution effects in human liver pyruvate kinase (hLPYK), including altered allosteric coupling. To facilitate a comparison of which characteristics determine when a nonconserved position does vs. does not contribute to function, the goal of the current work was to identify neutral positions in hLPYK. However, existing hLPYK data showed that three features commonly associated with neutral positions - high sequence entropy, high surface exposure, and alanine scanning - lacked the sensitivity needed to guide experimental studies. We used multiple evolutionary patterns identified in a sequence alignment of the PYK family to identify which positions were least patterned, reasoning that these were most likely to be neutral. Nine positions were tested with a total of 117 amino acid substitutions. Although exploring all potential functions is not feasible for any protein, five parameters associated with substrate/effector affinities and allosteric coupling were measured for hLPYK variants. For each position, the aggregate functional outcomes of all variants were used to quantify a "neutrality" score. Three positions showed perfect neutral scores for all five parameters. Furthermore, the nine positions showed larger neutral scores than 17 positions located near allosteric binding sites. Thus, our strategy successfully enriched the dataset for positions with neutral and modest substitutions.

biochemistry↗

bioRxiv2017

Protein-protein interactions with fructose-1-kinase alter function of the central Escherichia coli transcription regulator, Cra

In E. coli, the master transcription regulator Cra regulates >100 genes in central metabolism by binding upstream DNA operator sequences. Genes encoding glycolytic enzymes are repressed, whereas those for gluconeogenesis and the citric acid cycle are activated. Cra-DNA binding is allosterically diminished by binding to either fructose-1-phosphate (F-1-P, generated upon fructose import) or fructose-1,6-bisphosphate (F-1,6-BP). F-1,6-BP is generated from F-1-P by the enzyme fructose-1-kinase (FruK) or from other sugars and is a key intermediate in glycolysis. Here, we report that Cra directly interacts with FruK to form a tight protein-protein complex. Further, growth assays with a fruK knockout strain show that FruK has a broader role in metabolism than its known role in fructose catabolism. Biochemical experiments show that F-1,6-BP binding enhances either the Cra/FruK interaction and/or CRA binding to DNA and that FruK can catalyze the reverse reaction of F-1,6-BP to F-1-P. Results were used to propose a model in which the Cra-FruK complex enhances activation of gluconeogenic genes. Finally, since FruK itself is repressed by Cra, these newly-reported events add layers to the dynamic regulation of E. coli central metabolism that occur in response to changing nutrients.

microbiology↗