An intrinsically disordered transcription activation domain increases the DNA binding affinity and reduces the specificity of NFκB p50/RelA.

Many transcription factors contain intrinsically disordered transcription activation domains (TADs), which mediate interactions with coactivators to activate transcription. Historically, DNA-binding domains and TADs have been considered as modular units, but recent studies have shown that TADs can influence DNA binding. Whether these results can be generalized to more TADs is not clear. Here, we biophysically characterized the NFκB p50/RelA heterodimer including the RelA TAD and investigated the TAD's influence on NFκB-DNA interactions. In solution, we show the RelA TAD is disordered but compact, with helical tendency in two regions that interact with coactivators. We determined that the presence of the TAD increased the stoichiometry of NFκB-DNA complexes containing promoter DNA sequences with tandem κB recognition motifs by promoting the binding of NFκB dimers in excess of the number of κB sites. In addition, we measured the binding affinity of p50/RelA for DNA containing tandem κB sites and single κB sites. While the presence of the TAD enhanced the binding affinity of p50/RelA for all κB sequences tested, it also increased the affinity for nonspecific DNA sequences by over 10-fold, leading to an overall decrease in specificity for κB DNA sequences. In contrast, previous studies have generally reported that TADs decrease DNA-binding affinity and increase sequence specificity. Our results reveal a novel function of the RelA TAD in promoting binding to nonconsensus DNA, which sheds light on previous observations of extensive nonconsensus DNA binding by NFκB in vivo in response to strong inflammatory signals.

Protein Footprinting, Conformational Dynamics, and Core Interface-Adjacent Neutralization "Hotspots" in the SARS-CoV-2 Spike Protein Receptor Binding Domain/Human ACE2 Interaction.

The novel severe respiratory syndrome-like coronavirus (SARS-CoV-2) causes COVID-19 in humans and is responsible for one of the most destructive pandemics of the last century. At the root of SARS-CoV infection is the interaction between the viral spike protein and the human angiotensin converting enzyme 2 protein, which allows the virus to gain entry into host cells through endocytosis. In this work, we apply hydrogen-deuterium exchange mass spectrometry (HDX-MS) to provide a detailed view of the functional footprint and conformational dynamics associated with this interaction. Our results broadly agree with the binding interface derived from high resolution X-ray crystal structure data but also provide insights into shifts in structure and dynamics that accompany complexation, including some that occur immediately outside of the core binding interface. We propose that dampening of these "binding-site adjacent" dynamic shifts could represent a mechanism for neutralizing activity in a multitude of spike protein-targeted mAbs that have been found to specifically bind these "peripheral" sites. Our results highlight the unique capacity of HDX-MS to detect potential neutralization "hotspots" outside of the core binding interfaces defined by high resolution structural data.

HDX-MS: An Analytical Tool to Capture Protein Motion in Action.

Virtually all protein functions in the cell, including pathogenic processes, require coordinated motion of atoms or domains, i.e., conformational dynamics. Understanding protein dynamics is therefore critical both for drug development and to learn about the underlying molecular causes of many diseases. Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS) provides valuable information about protein dynamics, which is highly complementary to the static picture provided by conventional high-resolution structural tools (i.e., X-ray crystallography and structural NMR). The amount of protein required to carry out HDX-MS experiments is a fraction of the amount required by alternative biophysical techniques, which are also usually lower resolution. Use of HDX-MS is growing quickly both in industry and academia, and it has been successfully used in numerous drug and vaccine development efforts, with important roles in understanding allosteric effects and mapping binding sites.

Prediction of the presence of a seventh ankyrin repeat in IκBε from homology modeling combined with hydrogen-deuterium exchange mass spectrometry (HDX-MS).

The ankyrin repeat (AR) structure is a common protein-protein interaction motif and ankyrin repeat proteins comprise a vast family across a large array of different taxa. Natural AR proteins adopt a conserved fold comprised of several repeats with the N- and C-terminal repeats generally being of more divergent sequences. Obtaining experimental crystal structures for natural ankyrin repeat domains (ARD) can be difficult and often requires complexation with a binding partner. Homology modeling is an attractive method for creating a model of AR proteins due to the highly conserved fold; however, modeling the divergent N- and C-terminal "capping" repeats remains a challenge. We show here that amide hydrogen/deuterium exchange mass spectrometry (HDX-MS), which reports on the presence of secondary structural elements and "foldedness," can aid in the refinement and selection of AR protein homology models when multiple templates are identified with variations between them localizing to these terminal repeats. We report a homology model for the AR protein IκBε from three different templates and use HDX-MS to establish the presence of a seventh AR at the C-terminus identified by only one of the three templates used for modeling.

RelA-Containing NFκB Dimers Have Strikingly Different DNA-Binding Cavities in the Absence of DNA.

The main nuclear factor kappa B transcription factor family members RelA-p50 heterodimer and RelA homodimer have different biological functions and show different transcriptional activation profiles. To investigate whether the two family members adopt a similar conformation in their free states, we performed hydrogen-deuterium exchange mass spectrometry, all-atom molecular dynamics simulations, and stopped-flow binding kinetics experiments. Surprisingly, the N-terminal DNA-binding domains adopt an open conformation in RelA-p50 but a closed conformation in RelA homodimer. Both hydrogen-deuterium exchange mass spectrometry and molecular dynamics simulations indicate the formation of an interface between the N-terminal DNA-binding domains only in the RelA homodimer. Such an interface would be expected to impede DNA binding, and stopped-flow binding kinetics show that association of DNA is slower for the homodimer as compared to the heterodimer. Our results show that the DNA-binding cavity in the RelA-p50 heterodimer is open for DNA binding, whereas in the RelA homodimer, it is occluded.

Site-Specific Fluorescence Depolarization Kinetics Distinguishes the Amyloid Folds Responsible for Distinct Yeast Prion Strains.

The prion determinant of a yeast prion protein, Sup35NM, assembles into ß-rich amyloid fibrils that switch the nonprion [psi-] state to the prion [PSI+] state of yeast. Previous studies showed that two distinct forms of amyloids (Sc4 and Sc37), generated in vitro at two different temperatures (4 and 37 °C), recapitulate the strain phenomenon in Saccharomyces cerevisiae. Sc4 demonstrates a strong [PSI+] phenotype, whereas Sc37 shows a weak phenotype. To discern the residue-specific structural and dynamical attributes associated with the amyloids that display strain diversity, we took advantage of the nonoccurrence of tryptophan (Trp) in the NM-domain and created 18 single-Trp variants spanning the entire polypeptide length. The fluorescence readouts from these locations reported the site-specific structural details in Sc4 and Sc37 fibrils. Highly sensitive picosecond fluorescence depolarization measurements at these positions allowed a conformational mobility map to be constructed. Nearly all of the residue positions demonstrated higher local flexibility in Sc4 amyloid, which exhibits a strong phenotype. The differences in the amplitude of local mobility were more pronounced at the two end segments of the N-domain than in the central region. The M-domain is partially exposed and exhibits a higher amplitude of local mobility, indicating a lower degree of chain packing in the amyloid state, as well as a higher mobility in the Sc4 state compared to the Sc37 state. The altered local conformational dynamics in these two distinct amyloid states provide molecular insights into the varied fragility and severing efficiency that govern the inheritance patterns of strong and weak prion strains.

Stepwise unfolding of human ß2-microglobulin into a disordered amyloidogenic precursor at low pH.

Amyloid fibril formation by human ß2-microglobulin (ß2m) is associated with dialysis-related amyloidosis. In order to understand the mechanism of protein misfolding, it is important to characterize the nature and properties of various intermediates formed during protein unfolding. In this work, we studied the effect of pH change on the unfolding of ß2m using a range of spectroscopic readouts. In order to investigate the local structural changes, we created single tryptophan (W60 and W95) mutants of ß2m. The equilibrium results suggested that in the acid-unfolded state of ß2m at pH 2.5, the W60 residue attains non-native local structure whereas the W95 residue becomes more exposed. Our stopped-flow kinetic data revealed that ß2m undergoes unfolding in a stepwise manner. Initial unfolding of ß2m involves non-uniform protein expansion with the unpacking of tertiary structure and significant core solvation while maintaining a native-like structure around residue W95. The resolved-phase of unfolding exhibits a timescale of ~500 ms that describes the transition from the native-like swollen intermediate to an acid-induced disordered state. Taken together, our results demonstrate that ß2m has a complex pH-induced unfolding mechanism yielding a disordered amyloidogenic precursor comprising both exposed and buried segments.

Direct Observation of the Intrinsic Backbone Torsional Mobility of Disordered Proteins.

The fundamental backbone dynamics of unfolded proteins arising due to intrinsic ϕ-ψ dihedral angle fluctuations dictate the course of protein folding, binding, assembly, and function. These internal fluctuations are also critical for protein misfolding associated with a range of human diseases. However, direct observation and unambiguous assignment of this inherent dynamics in chemically denatured proteins is extremely challenging due to various experimental limitations. To directly map the backbone torsional mobility in the ϕ-ψ dihedral angle space, we used a model intrinsically disordered protein, namely, α-synuclein, that adopts an expanded state under native conditions. We took advantage of nonoccurrence of tryptophan in α-synuclein and created a number of single-tryptophan variants encompassing the entire polypeptide chain. We then utilized highly sensitive picosecond time-resolved fluorescence depolarization measurements that allowed us to discern the site-specific torsional relaxation at a low protein concentration under physiological conditions. For all the locations, the depolarization kinetics exhibited two well-separated rotational-correlation-time components. The shorter, subnanosecond component arises due to the local mobility of the indole side chain, whereas the longer rotational-correlation-time component (1.37 ± 0.15 ns), independent of global tumbling, represents a characteristic timescale for short-range conformational exchange in the ϕ-ψ dihedral space. This correlation time represents an intrinsic timescale for torsional relaxation and is independent of position, which is expected for an extended polypeptide chain having little or no propensity to form persistent structures. We were also able to capture this intrinsic timescale at the N-terminal unstructured domain of the prion protein. Our estimated timescale of the segmental mobility is similar to that of unfolded proteins studied by nuclear magnetic resonance in conjunction with molecular dynamics simulations. Our results have broader implications for a diverse range of functionally and pathologically important intrinsically disordered proteins and disordered regions.

Characterization of Salt-Induced Oligomerization of Human ß2-Microglobulin at Low pH.

Misfolding and amyloid aggregation of human ß2-microglobulin (ß2m) have been linked to dialysis-related amyloidosis. Previous studies have shown that in the presence of different salt concentrations and at pH 2.5, ß2m assembles into aggregates with distinct morphologies. However, the structural and mechanistic details of the aggregation of ß2m, giving rise to different morphologies, are poorly understood. In this work, we have extensively characterized the salt-induced oligomers of the acid-unfolded state of ß2m using an array of biophysical tools including steady-state and time-resolved fluorescence, circular dichroism, dynamic light scattering, and atomic force microscopy imaging. Fluorescence studies using the oligomer-sensitive molecular rotor, 4-(dicyanovinyl)-julolidine, in conjunction with the light scattering and cross-linking assay indicated that at low salt (NaCl) concentrations ß2m exists as a disordered monomer, capable of transforming into ordered amyloid. In the presence of higher concentrations of salt, ß2m aggregates into a larger oligomeric species that does not appear to transform into amyloid fibrils. Site-specific fluorescence experiments using single Trp variants of ß2m revealed that the middle region of the protein is incorporated into these oligomers, whereas the C-terminal segment is highly exposed to bulk water. Additionally, stopped-flow kinetic experiments indicated that the formation of hydrophobic core and oligomerization occur concomitantly. Our results revealed the distinct pathways by which ß2m assembles into oligomers and fibrils.

Molecular characterization of oxidative stress-inducible LipD of Mycobacterium tuberculosis H37Rv.

The Mycobacterium tuberculosis has developed intricate strategies to evade the killing of microorganism and support its survival in phagocytes. The genome sequence of bacterium revealed the presence of several genes for lypolytic enzymes. Rv1923 gene, a member of Lip family in M. tuberculosis demonstrated the least sequence similarity with its counterpart in non-pathogenic strain M. smegmatis. The expression of Rv1923 gene (LipD) was not observed in in vitro growing cultures of M. tuberculosis H37Ra while an upregulation of transcription of Rv1923 gene was noticed in oxidative conditions. For detailed characterization of LipD enzyme the Rv1923 gene was cloned in pQE30-UA vector and expressed in E. coli M15 cells. LipD was purified from inclusion bodies and refolded with nearly 40 % protein yield. The specific activity of enzyme was calculated to be 16 U/mg with pNP-palmitate as a preferred substrate. Kinetic analysis showed K(m) 0.645 mM and V(max) 24.75 U/ml with pNP-palmitate. Ser-102, Asp-342, and His-369, predicted as the members of the catalytic triad, were confirmed by mutagenesis. Mutagenesis studies revealed that catalytic serine residues located in ß-lactamase motifs (S-X-X-K) were responsible for lipolytic activity. Secondary structure analysis by CD spectroscopy demonstrated the presence of α helices and ß sheets in the canonical structure of LipD. The enzyme was stable up to 50 °C and was active even at pH 6.0. The expression of enzyme under stress conditions and its activity and stability at high temperature and low pH suggested the possible role of LipD in the survival of mycobacterium in macrophage compartment.

Characterization of an acid inducible lipase Rv3203 from Mycobacterium tuberculosis H37Rv.

The Rv3203 (LipV) of Mycobacterium tuberculosis (Mtb) H37Rv, is annotated as a member of Lip family based on the presence of characteristic consensus esterase motif 'GXSXG'. In vitro culture studies of Mtb H37Ra indicated that expression of Rv3203 gene was up-regulated during acidic stress as compared to normal whereas no expression was observed under nutrient and oxidative stress conditions. Therefore, detailed characterization of Rv3203 was done by gene cloning and its further expression and purification as his-tagged protein in microbial expression system. The enzyme was purified to homogeneity by affinity chromatography. It demonstrated broad substrate specificity and preferentially hydrolyzed p-nitrophenyl myristate. The purified enzyme demonstrated an optimum activity at pH 8.0 and temperature 50 °C. The specific activity, K m and V max of enzyme was determined to be 21.29 U mg(-1) protein, 714.28 µM and 62.5 µmol ml(-1) min(-1), respectively. The pH stability assay and circular dichroism spectroscopic analysis revealed that Rv3203 protein is more stable in acidic condition. Tetrahydrolipstatin, a specific lipase inhibitor and RHC80267, a diacylglycerol lipase inhibitor abolished the activity of this enzyme. The catalytic triad residues were determined to be Ser50, Asp180 and His203 residues by site-directed mutagenesis.

Dynamics and dimension of an amyloidogenic disordered state of human ß(2)-microglobulin.

Human ß2-microglobulin (ß2m) aggregation is implicated in dialysis-related amyloidosis. Previously, it has been shown that ß2m adopts an ensemble of partially unfolded states at low pH. Here we provide detailed structural and dynamical insights into the acid unfolded and yet compact state of ß2m at pH 2.5 using a host of fluorescence spectroscopic tools. These tools allowed us to investigate protein conformational dynamics at low micromolar protein concentrations in an amyloid-forming condition. Our equilibrium fluorescence data in combination with circular dichroism data provide support in favor of progressive structural dissolution of ß2m with lowering pH. The acid unfolded intermediate at pH 2.5 has high 8-anilinonaphthalene, 1-sulfonic acid (ANS)-binding affinity and is devoid of significant secondary structural elements. Using fluorescence lifetime measurements, we have been able to monitor the conformational transition during the pH transition from the native to the compact disordered state. Additionally, using time-resolved fluorescence anisotropy measurements, we have been able to distinguish this compact disordered state from the canonical denatured state of the protein by identifying unique dynamic signatures pertaining to the segmental chain mobility. Taken together, our results demonstrate that ß2m at pH 2.5 adopts a compact noncanonical unfolded state resembling a collapsed premolten globule state. Additionally, our stopped-flow fluorescence kinetics results provide mechanistic insights into the formation of a compact disordered state from the native form.

Nanoscale Fluorescence Imaging of Single Amyloid Fibrils.

Amyloid formation is implicated in a variety of human diseases. It is important to perform high-resolution optical imaging of individual amyloid fibrils to delineate the structural basis of supramolecular protein assembly. However, amyloid fibrils do not lend themselves to the conventional microscopic resolution, which is hindered by the diffraction limit. Here we show super-resolution fluorescence imaging of fluorescently stained amyloid fibrils derived from disease-associated human ß2-microglobulin using near-field scanning fluorescence microscopy. Using this technique, we were able to resolve the fibrils that were spatially separated by ∼75 nm. We have also been able to interrogate individual fibrils in a fibril-by-fibril manner by simultaneously monitoring both nanoscale topography and fluorescence brightness along the length of the fibrils. This method holds promise to detect conformational distributions and heterogeneity that are believed to correlate with the supramolecular packing of misfolded proteins within the fibrils in a diverse conformationally enciphered prion strains and amyloid polymorphs.