Protein dynamics─the fluctuating nature of protein structure once described by Gregorio Weber as "kicking and screaming"─is understood to be an intrinsic feature of proteins and their function. Yet it is often difficult to pin down exactly how those dynamics assist function. Allosteric regulation is a widespread protein function that was once seen to operate solely through conformational change. Over the last two decades, a series of experimental studies has shown that thermally activated, rapid-time scale dynamics can underlie allosteric ligand binding cooperativity, even in the absence of conformational change. This concept is known as "dynamic allostery", in which localized dynamics represent conformational entropy that can effectively serve as a set of thermodynamic "nano-levers". Here, we review these studies and their collective finding: that changes in the amplitudes of picosecond-nanosecond timescale side-chain dynamics can exert a large entropic driving force in protein binding events. The studies require NMR relaxation measurements of methyl "order parameters" (O2axis). We focus on the recent example from Sgt2, a chaperone in yeast's guided tail-anchoring protein pathway. Sgt2 harbors an intrinsically disordered C-terminal tail that allosterically enhances side-chain dynamics in other domains, which in turn abrogates binding to partner Get4/5. Motivated by this example, order parameters are explained in simple terms and discussed empirically to raise confidence in them as meaningful reporters of local motion. Specific studies are highlighted to show that different proteins utilize distinct dynamic strategies for allosteric coupling. Finally, the surprising role of disordered tails in controlling dynamic allostery is discussed.

Flavin mononucleotide (FMN)-dependent nitroreductases offer a mild and selective route to reduce nitroaromatic compounds, yet these typically fail to generate the corresponding amines. This incomplete transformation has been attributed to the two-electron redox potential of FMN. To test this hypothesis, the major nitroreductase NfsA from Escherichia coli was reconstituted with a series of FMN variants spanning midpoint potentials of -215 to -307 mV. Product profiles were examined with four substrates covering a range of electron affinities (nitrofurazone, 1,3-dinitrobenzene, 4-nitroacetophenone, and nitrobenzene), and all combinations of enzymes and substrates were found to yield only the hydroxylamine products. No amines were detected under any condition, and as a confirmation, 4-hydroxylaminacetophenone was shown to be inert to treatment with reduced nicotinamide adenine dinucleotide phosphate and NfsA reconstituted with a low-potential FMN variant (-295 mV). The inability of NsfA to generate amines is consequently not a function of the reducing potential. However, this is a determinant of the catalytic efficiency. The kcat for nitroacetophenone turnover decreased almost 240-fold for NfsA containing an FMN variant with an Em of -307 mV relative to that containing native FMN (-215 mV). 1,3-Dinitrobenzene experienced the smallest decrease of 52-fold in the same comparison. Redox tuning of NfsA can therefore be detrimental to catalytic efficiency and fails to generate the desired amine products. A renewed focus on active site properties is recommended for engineering new catalysts to promote nitroaromatic to arylamine conversion.

ISGylation is a ubiquitin-like post-translational modification that plays a central role in innate immune signaling. Conjugation of interferon-stimulated gene 15 (ISG15) to target proteins is initiated by the E1 enzyme Uba7, transferred to the E2 enzyme UbcH8, and completed by an E3 ligase. Specificity in this cascade is mediated by the ubiquitin-fold domain (UFD) of Uba7, yet the structural and mechanistic basis of E1-E2 recognition remains poorly defined. Here, we present the solution NMR structure and functional characterization of a human Uba7-UFD. NMR chemical shift perturbation experiments combined with site-directed mutagenesis delineate the UbcH8 interaction surface and identify residues critical for E1-E2 binding. The Uba7-UFD adopts a conserved ubiquitin-fold architecture but exhibits conformational flexibility in the unbound state. 15N relaxation measurements show a globally well-folded domain with localized ps-ns time scale dynamics within the β2/β4 E2 binding surface and the acidic loop spanning residues 996-1008. Upon UbcH8 binding, relaxation parameters shift toward those expected for a larger effective molecular size, accompanied by an increased residue-specific heterogeneity at the interface, consistent with binding-coupled changes in local mobility. Mutational analysis identifies C996 as being essential for UFD structural integrity and binding competence. Moreover, targeted alterations in the length and flexibility of the adjacent acidic loop strongly impair UbcH8 binding, demonstrating that the loop architecture is a critical determinant of efficient E2 recruitment. Together, these results provide a structural and dynamic framework for understanding E2 enzyme selection in the ISGylation pathway and highlight the role of UFD conformational dynamics in the E1-E2 complex formation.

Fumarate-adding enzymes (FAE) are a subset of the glycyl radical enzyme superfamily involved in anaerobic hydrocarbon degradation. Benzylsuccinate synthase (BSS) catalyzes the enantiospecific formation of R-benzylsuccinate from toluene and fumarate, initiating anaerobic toluene degradation. In this paper, we present a detailed theoretical study of the reaction mechanism using classical molecular dynamics and multiscale modeling (QM/MM). We describe the potential energy surface of the reaction and confirm the previously postulated mechanism. However, the multiscale character of our model allowed us to elucidate the origins of several experimentally observed catalytic phenomena, such as the inversion of the benzylic carbon configuration upon C-C bond formation and the syn addition of the abstracted H atom back to the benzylsuccinyl radical. The obtained model is supported by microkinetic analysis and was able to explain and quantitatively predict the strict R-enantioselectivity of BSS, which is enforced predominantly by the dynamic kinetic behavior of toluene in the active site, leading to over 40-times faster production of the R-enantiomer, not by the binding orientation of the fumarate. Our study contributes to the elucidation of the catalytic processes catalyzed by BSS and its role in the bioremediation of hydrocarbon pollutants.

Protein unfolded states are heterogeneous but can manifest local and long-range order. Replacement of side chains through site-directed mutagenesis is a common method to manipulate the unfolded state and elucidate its role in the folding process. Modification of the protein backbone represents a less explored complementary approach with the potential to elicit dramatic changes in conformational preferences from minimal chemical alteration. Prior work has shown backbone modification can affect unfolded ensembles as well as intrinsically disordered sequences. Here, we show that it can be used to rationally tune structural characteristics of the unfolded state of a folded protein. Using the GCN4 leucine zipper as a host, canonical α-residues throughout the chain are individually replaced by β3 or Cα-Me-α analogues. The former modification enhances conformational freedom, the latter restricts it, and both retain the side chain at the substitution site. Characterization by circular dichroism and X-ray crystallography shows that the variants adopt folded structures identical to the prototype. Thermal and thermodynamic stability vary in complex ways with the context and nature of backbone modification; however, a uniform relationship is observed between substitution type and the sensitivity of folding free energy to chemical denaturant. This finding suggests systematic changes in solvent-accessible surface area of the unfolded ensemble among isomeric proteins differing only in the position of a single CH2 group. Collectively, these results demonstrate a platform for predictably tuning the properties of the unfolded state through minimal chemical modification, enabling new avenues for fundamental research on folding behavior of proteins as well as protein mimetics.

Nesprin-2 and its paralog Nesprin-1 are subunits of LINC complexes that are essential for brain development. To position the nucleus for neuronal migration, Nesprin-2 interacts with the motors kinesin-1 and dynein, which are recruited by the adapter Bicaudal D2 (BicD2), but the molecular details of these interactions are elusive. Here, structural models of minimal Nesprin-2/BicD2 complexes with 1:2 and 2:2 stoichiometry were predicted using AlphaFold and experimentally validated by mutagenesis, binding assays, and single-molecule biophysical studies. The core of the binding site is formed by spectrin repeats of Nesprin-2, which form an α-helical bundle with BicD2 that is structurally distinct from the Rab6/BicD2 and Nup358/BicD2 complexes. Such structural differences could fine-tune the motility of associated dynein and kinesin-1 motors for these transport pathways. Furthermore, the Nesprin-2 fragment interacts with full-length BicD2 and activates dynein/dynactin/BicD2 complexes for processive motility, suggesting that no additional components are required to reconstitute this transport pathway. Interestingly, either one or two Nesprin-2 molecules can bind to a BicD2 dimer and activate BicD2/dynein/dynactin complexes for processive motion, resulting in similar speed and run lengths. The BicD2/dynein binding site is spatially close but does not overlap with the kinesin-1 recruitment site, thus both motors may interact with Nesprin-2 simultaneously. Several mutations of Nesprin-1 and 2 that cause Emery-Dreifuss muscular dystrophy are found in the motor-recruiting domain and may alter interactions with kinesin-1 and BicD2/dynein, consistent with the abnormally positioned nuclei found in patients with this disease.

DNA G-quadruplexes (G4s) are key structural features in chromatin that are important to genome function. G4s have an apparent capacity to recruit a wide variety of proteins, including chromatin remodelers, yet the molecular basis and biophysical principles governing these interactions remain poorly understood. Here, we sought to build insights into the interactions of chromatin remodeler SMARCA4 with G4s using a biophysical approach. We found that SMARCA4 selectively recognizes the G4 structure over duplex and single-stranded DNA. SMARCA4 binds a wide range of G4s with different topologies and loop lengths with similar low nanomolar affinities. SMARCA4 was also observed to have a longer residency time on the G4 structure compared to that of other known protein-DNA interactions. We also found that the D1 (DExx-c) helicase domain of SMARCA4, which is important for tethering SMARCA4 to chromatinized DNA, was the predominant binding domain for G4 recognition. Our findings reveal new insights into how G4s interact with proteins, which may have important implications for understanding G4-mediated genome mechanisms.

Collectively known as one-carbon metabolism (OCM), both the folate and methionine cycles are highly regulated to meet cellular demands. These cycles are key in the production and recycling of methyl groups to be used in many essential cellular processes such as the production of nucleotides, as well as S-adenosyl-l-methionine (SAM) the global methyl donor for DNA, RNA, and post translational modifications. Within the folate cycle, 5,10-methylenetetrahydrofolate is the main species through which methyl groups enter OCM. Therefore, 5,10-methylenetetrahydrofolate reductase (MTHFR), which reduces 5,10-methylenetetrahydrofolate into 5-methyltetrahydrofolate, is the central enzyme that directs methyl groups for use within the methionine cycle. MTHFR is an enzyme found in all domains of life, but unlike in prokaryotes, eukaryotic MTHFR activity is highly regulated by the level of SAM, to balance the one-carbon needs of the cell. In this perspective, we review the catalytic mechanism of MTHFR, evolutionary differences, and the regulatory mechanisms that have evolved to alter its activity. We also discuss recent structural findings that reveal a unique mechanism for inactivation by SAM as a feedback loop and its consequences for understanding inherited MTHFR deficiency.