Biology subjects

McKnight, S. L.

Publications and source records attributed to McKnight, S. L..

5 recordsLinked to original sources

bioRxiv2021

Functional Anatomy of the TDP-43 Redox Sensor

TAR binding protein 43 (TDP-43) is an RNA binding protein that assists in the maturation, export and sub-cellular localization of mRNA. The carboxyl terminal 153 residues of TDP-43 are of low sequence complexity and allow for self-association of the protein in a manner leading to its phase separation from an aqueous environment. These interactions assist TDP-43 in forming cytoplasmic RNA granules involved in the transport of mRNA for localized translation. Self-association of the TDP-43 low complexity (LC) domain is facilitated by a region of twenty five residues that are of extreme evolutionary conservation. The molecular basis for self-adherence of the protein through this region has been illuminated by a combination of structural and biochemical studies, allowing definition of a morphologically specific cross-{beta} structure predicted to be weakly assembled by main chain hydrogen bonds. In this study we have investigated the importance of individual, Pauling hydrogen bonds hypothesized to facilitate self-adherence of the TDP-43 LC domain.

biochemistry↗

bioRxiv2021

The Low Complexity Domain of the FUS RNA Binding Protein Self-assembles via the Mutually Exclusive Use of Two Distinct Cross-β Cores

The low complexity (LC) domain of the fused in sarcoma (FUS) RNA binding protein self-associates in a manner causing phase separation from an aqueous environment. Incubation of the FUS LC domain under physiologically normal conditions of salt and pH leads to rapid formation of liquid-like droplets that mature into a gel-like state. Both examples of phase separation have enabled reductionist biochemical assays allowing discovery of an N-terminal region of 56 residues that assembles into a labile, cross-{beta} structure. Here we provide evidence of a non-overlapping, C-terminal region of the FUS LC domain that also forms specific cross-{beta} interactions. We propose that biologic function of the FUS LC domain may operate via the mutually exclusive use of these N- and C-terminal cross-{beta} cores. Neurodegenerative disease-causing mutations in the FUS LC domain are shown to imbalance the two cross-{beta} cores, offering an unanticipated concept of LC domain function and dysfunction. Significance StatementSingle amino acid changes causative of neurologic disease often map to the cross-{beta} forming regions of low complexity (LC) domains. All such mutations studied to date lead to enhanced avidity of cross-{beta} interactions. The LC domain of the fused in sarcoma (FUS) RNA binding protein contains three different regions that are capable of forming labile cross-{beta} interactions. Here we describe the perplexing effect of amyotrophic lateral sclerosis (ALS)-causing mutations localized to the LC domain of FUS to substantially weaken its ability to form one of its three cross-{beta} interactions. An understanding of how these mutations abet uncontrolled polymerization of the FUS LC domain may represent an important clue as to how LC domains achieve their proper biological function.

biochemistry↗

bioRxiv2020

Dynamic structural order of a low complexity domain facilitates assembly of intermediate filaments

The coiled-coil domains of intermediate filament (IF) proteins are flanked by regions of low sequence complexity. Whereas IF coiled-coil domains assume dimeric and tetrameric conformations on their own, maturation of eight tetramers into cylindrical IFs is dependent upon either "head" or "tail" domains of low sequence complexity. Here we confirm that the tail domain required for assembly of Drosophila Tm1 IFs functions by forming labile cross-{beta} interactions. These interactions are seen in polymers made from the tail domain alone as well as assembled IFs formed by the intact Tm1 protein. The ability to visualize such interactions in situ within the context of a discrete cellular assembly lends support to the concept that equivalent interactions may be used in organizing other dynamic aspects of cell morphology. One Sentence SummaryA new form of protein folding that interconverts between the structured and unstructured states controls assembly of intermediate filaments.

biochemistry↗

bioRxiv2020

Transiently structured head domains control intermediate filament assembly

Low complexity (LC) head domains 92 and 108 residues in length are, respectfully, required for assembly of neurofilament light (NFL) and desmin intermediate filaments (IFs). As studied in isolation, these IF head domains interconvert between states of conformational disorder and labile, {beta}-strand-enriched polymers. Solid state nuclear magnetic resonance (ss-NMR) spectroscopic studies of NFL and desmin head domain polymers reveal spectral patterns consistent with structural order. A combination of intein chemistry and segmental isotope labeling allowed preparation of fully assembled NFL and desmin IFs that could also be studied by ss-NMR. Assembled IFs revealed spectra overlapping with those observed for {beta}-strand-enriched polymers formed from the isolated NFL and desmin head domains. Phosphorylation and disease causing mutations reciprocally alter NFL and desmin head domain self-association, yet commonly impede IF assembly. These observations show how facultative structural assembly of LC domains via labile, {beta}-strand-enriched self-interactions may broadly influence cell morphology.

biochemistry↗

bioRxiv2019

Redox-mediated regulation of a labile, evolutionarily conserved cross-β structure formed by the TDP43 low complexity domain

An evolutionarily conserved low complexity (LC) domain is found within a 152 residue segment localized to the carboxyl-terminal region of the TDP43 RNA-binding protein. This TDP43 LC domain contains ten conserved methionine residues. Self-association of this domain leads to the formation of liquid-like droplets composed of labile, cross-{beta} polymers. Exposure of polymers to low concentrations of H2O2 leads to a phenomenon of droplet melting that can be reversed upon exposure of the oxidized protein to the MsrA and MsrB methionine sulfoxide reductase enzymes, thioredoxin, thioredoxin reductase and NADPH. Morphological features of the cross-{beta} polymers were revealed by a method of H2O2-mediated footprinting. Similar TDP43 LC domain footprints were observed in highly polymerized, hydrogel samples, liquid-like droplet samples, and living cells. The ability of H2O2 to impede cross-{beta} polymerization was abrogated by a prominent ALS-causing mutation that changes methionine residue 337 to valine. These observations offer potentially useful insight into the biological role of TDP43 in facilitating synapse-localized translation, as well as aberrant aggregation of the protein in neurodegenerative disease.

biochemistry↗