Structural Control of Nonnative Ligand Binding in Engineered Mutants of Phosphoenolpyruvate Carboxykinase.

Protein engineering to alter recognition underlying ligand binding and activity has enormous potential. Here, ligand binding for Escherichia coli phosphoenolpyruvate carboxykinase (PEPCK), which converts oxaloacetate into CO2 and phosphoenolpyruvate as the first committed step in gluconeogenesis, was engineered to accommodate alternative ligands as an exemplary system with structural information. From our identification of bicarbonate binding in the PEPCK active site at the supposed CO2 binding site, we probed binding of nonnative ligands with three oxygen atoms arranged to resemble the bicarbonate geometry. Crystal structures of PEPCK and point mutants with bound nonnative ligands thiosulfate and methanesulfonate along with strained ATP and reoriented oxaloacetate intermediates and unexpected bicarbonate were determined and analyzed. The mutations successfully altered the bound ligand position and orientation and its specificity: mutated PEPCKs bound either thiosulfate or methanesulfonate but never both. Computational calculations predicted a methanesulfonate binding mutant and revealed that release of the active site ordered solvent exerts a strong influence on ligand binding. Besides nonnative ligand binding, one mutant altered the Mn2+ coordination sphere: instead of the canonical octahedral ligand arrangement, the mutant in question had an only five-coordinate arrangement. From this work, critical features of ligand binding, position, and metal ion cofactor geometry required for all downstream events can be engineered with small numbers of mutations to provide insights into fundamental underpinnings of protein-ligand recognition. Through structural and computational knowledge, the combination of designed and random mutations aids in the robust design of predetermined changes to ligand binding and activity to engineer protein function.

High Resolution Distance Distributions Determined by X-Ray and Neutron Scattering.

Measuring distances within or between macromolecules is necessary to understand the chemistry that biological systems uniquely enable. In performing their chemistry, biological macromolecules undergo structural changes over distances ranging from atomic to micrometer scales. X-ray and neutron scattering provide three key assets for tackling this challenge. First, they may be conducted on solutions where the macromolecules are free to sample the conformations that enable their chemistry. Second, there are few limitations on chemical environment for experiments. Third, the techniques can inform upon a wide range of distances at once. Thus scattering, particularly recorded at small angles (SAS), has been applied to a large variety of phenomenon. A challenge in interpreting scattering data is that the desired three dimensional distance information is averaged onto one dimension. Furthermore, the scales and variety of phenomenon interrogated have led to an assortment of functions that describe distances and changes thereof. Here we review scattering studies that characterize biological phenomenon at distances ranging from atomic to 50 nm. We also distinguish the distance distribution functions that are commonly used to describe results from these systems. With available X-ray and neutron scattering facilities, bringing the action that occurs at the atomic to the micrometer scale is now reasonably accessible. Notably, the combined distance and dynamic information recorded by SAS is frequently key to connecting structure to biological activity and to improve macromolecular design strategies and outcomes. We anticipate widespread utilization particularly in macromolecular engineering and time-resolved studies where many contrasting experiments are necessary for resolving chemical mechanisms through structural changes.

Charging of Proteins in Native Mass Spectrometry.

Factors that influence the charging of protein ions formed by electrospray ionization from aqueous solutions in which proteins have native structures and function were investigated. Protein ions ranging in molecular weight from 12.3 to 79.7 kDa and pI values from 5.4 to 9.6 were formed from different solutions and reacted with volatile bases of gas-phase basicities higher than that of ammonia in the cell of a Fourier-transform ion cyclotron resonance mass spectrometer. The charge-state distribution of cytochrome c ions formed from aqueous ammonium or potassium acetate is the same. Moreover, ions formed from these two solutions do not undergo proton transfer to 2-fluoropyridine, which is 8 kcal/mol more basic than ammonia. These results provide compelling evidence that proton transfer between ammonia and protein ions does not limit protein ion charge in native electrospray ionization. Both circular dichroism and ion mobility measurements indicate that there are differences in conformations of proteins in pure water and aqueous ammonium acetate, and these differences can account for the difference in the extent of charging and proton-transfer reactivities of protein ions formed from these solutions. The extent of proton transfer of the protein ions with higher gas-phase basicity bases trends with how closely the protein ions are charged to the value predicted by the Rayleigh limit for spherical water droplets approximately the same size as the proteins. These results indicate that droplet charge limits protein ion charge in native mass spectrometry and are consistent with these ions being formed by the charged residue mechanism. Graphical Abstract ᅟ.

Designing and defining dynamic protein cage nanoassemblies in solution.

Central challenges in the design of large and dynamic macromolecular assemblies for synthetic biology lie in developing effective methods for testing design strategies and their outcomes, including comprehensive assessments of solution behavior. We created and validated an advanced design of a 600-kDa protein homododecamer that self-assembles into a symmetric tetrahedral cage. The monomeric unit is composed of a trimerizing apex-forming domain genetically linked to an edge-forming dimerizing domain. Enhancing the crystallographic results, high-throughput small-angle x-ray scattering (SAXS) comprehensively contrasted our modifications under diverse solution conditions. To generate a phase diagram associating structure and assembly, we developed force plots that measure dissimilarity among multiple SAXS data sets. These new tools, which provided effective feedback on experimental constructs relative to design, have general applicability in analyzing the solution behavior of heterogeneous nanosystems and have been made available as a web-based application. Specifically, our results probed the influence of solution conditions and symmetry on stability and structural adaptability, identifying the dimeric interface as the weak point in the assembly. Force plots comparing SAXS data sets further reveal more complex and controllable behavior in solution than captured by our crystal structures. These methods for objectively and comprehensively comparing SAXS profiles for systems critically affected by solvent conditions and structural heterogeneity provide an enabling technology for advancing the design and bioengineering of nanoscale biological materials.

HU multimerization shift controls nucleoid compaction.

Molecular mechanisms controlling functional bacterial chromosome (nucleoid) compaction and organization are surprisingly enigmatic but partly depend on conserved, histone-like proteins HUαα and HUαß and their interactions that span the nanoscale and mesoscale from protein-DNA complexes to the bacterial chromosome and nucleoid structure. We determined the crystal structures of these chromosome-associated proteins in complex with native duplex DNA. Distinct DNA binding modes of HUαα and HUαß elucidate fundamental features of bacterial chromosome packing that regulate gene transcription. By combining crystal structures with solution x-ray scattering results, we determined architectures of HU-DNA nucleoproteins in solution under near-physiological conditions. These macromolecular conformations and interactions result in contraction at the cellular level based on in vivo imaging of native unlabeled nucleoid by soft x-ray tomography upon HUß and ectopic HUα38 expression. Structural characterization of charge-altered HUαα-DNA complexes reveals an HU molecular switch that is suitable for condensing nucleoid and reprogramming noninvasive Escherichia coli into an invasive form. Collective findings suggest that shifts between networking and cooperative and noncooperative DNA-dependent HU multimerization control DNA compaction and supercoiling independently of cellular topoisomerase activity. By integrating x-ray crystal structures, x-ray scattering, mutational tests, and x-ray imaging that span from protein-DNA complexes to the bacterial chromosome and nucleoid structure, we show that defined dynamic HU interaction networks can promote nucleoid reorganization and transcriptional regulation as efficient general microbial mechanisms to help synchronize genetic responses to cell cycle, changing environments, and pathogenesis.