Connecting sequence features within the disordered C-terminal linker of Bacillus subtilis FtsZ to functions and bacterial cell division.

Intrinsically disordered regions (IDRs) can function as autoregulators of folded enzymes to which they are tethered. One example is the bacterial cell division protein FtsZ. This includes a folded core and a C-terminal tail (CTT) that encompasses a poorly conserved, disordered C-terminal linker (CTL) and a well-conserved 17-residue C-terminal peptide (CT17). Sites for GTPase activity of FtsZs are formed at the interface between GTP binding sites and T7 loops on cores of adjacent subunits within dimers. Here, we explore the basis of autoregulatory functions of the CTT in Bacillus subtilis FtsZ (Bs-FtsZ). Molecular simulations show that the CT17 of Bs-FtsZ makes statistically significant CTL-mediated contacts with the T7 loop. Statistical coupling analysis of more than 1,000 sequences from FtsZ orthologs reveals clear covariation of the T7 loop and the CT17 with most of the core domain, whereas the CTL is under independent selection. Despite this, we discover the conservation of nonrandom sequence patterns within CTLs across orthologs. To test how the nonrandom patterns of CTLs mediate CTT-core interactions and modulate FtsZ functionalities, we designed Bs-FtsZ variants by altering the patterning of oppositely charged residues within the CTL. Such alterations disrupt the core-CTT interactions, lead to anomalous assembly and inefficient GTP hydrolysis in vitro and protein degradation, aberrant assembly, and disruption of cell division in vivo. Our findings suggest that viable CTLs in FtsZs are likely to be IDRs that encompass nonrandom, functionally relevant sequence patterns that also preserve three-way covariation of the CT17, the T7 loop, and core domain.

Making the Case for Disordered Proteins and Biomolecular Condensates in Bacteria.

Intrinsically disordered proteins/regions (IDPs/IDRs) contribute to a diverse array of molecular functions in eukaryotic systems. There is also growing recognition that membraneless biomolecular condensates, many of which are organized or regulated by IDPs/IDRs, can enable spatial and temporal regulation of complex biochemical reactions in eukaryotes. Motivated by these findings, we assess if (and how) membraneless biomolecular condensates and IDPs/IDRs are functionally involved in key cellular processes and molecular functions in bacteria. We summarize the conceptual underpinnings of condensate assembly and leverage these concepts by connecting them to recent findings that implicate specific types of condensates and IDPs/IDRs in important cellular level processes and molecular functions in bacterial systems.

Uncovering Non-random Binary Patterns Within Sequences of Intrinsically Disordered Proteins.

Sequence-ensemble relationships of intrinsically disordered proteins (IDPs) are governed by binary patterns such as the linear clustering or mixing of specific residues or residue types with respect to one another. To enable the discovery of potentially important, shared patterns across sequence families, we describe a computational method referred to as NARDINI for Non-random Arrangement of Residues in Disordered Regions Inferred using Numerical Intermixing. This work was partially motivated by the observation that parameters that are currently in use for describing different binary patterns are not interoperable across IDPs of different amino acid compositions and lengths. In NARDINI, we generate an ensemble of scrambled sequences to set up a composition-specific null model for the patterning parameters of interest. We then compute a series of pattern-specific z-scores to quantify how each pattern deviates from a null model for the IDP of interest. The z-scores help in identifying putative non-random linear sequence patterns within an IDP. We demonstrate the use of NARDINI derived z-scores by identifying sequence patterns in three well-studied IDP systems. We also demonstrate how NARDINI can be deployed to study archetypal IDPs across homologs and orthologs. Overall, NARDINI is likely to aid in designing novel IDPs with a view toward engineering new sequence-function relationships or uncovering cryptic ones. We further propose that the z-scores introduced here are likely to be useful for theoretical and computational descriptions of sequence-ensemble relationships across IDPs of different compositions and lengths.

Dissecting the Functional Contributions of the Intrinsically Disordered C-terminal Tail of Bacillus subtilis FtsZ.

FtsZ is a bacterial GTPase that is central to the spatial and temporal control of cell division. It is a filament-forming enzyme that encompasses a well-folded core domain and a disordered C-terminal tail (CTT). The CTT is essential for ensuring proper assembly of the cytokinetic ring, and its deletion leads to mis-localization of FtsZ, aberrant assembly, and cell death. In this work, we dissect the contributions of modules within the disordered CTT to assembly and enzymatic activity of Bacillus subtilis FtsZ (Bs-FtsZ). The CTT features a hypervariable C-terminal linker (CTL) and a conserved C-terminal peptide (CTP). Our in vitro studies show that the CTL weakens the driving forces for forming single-stranded active polymers and suppresses lateral associations of these polymers, whereas the CTP promotes the formation of alternative assemblies. Accordingly, in full-length Bs-FtsZ, the CTL acts as a spacer that spatially separates the CTP sticker from the core, thus ensuring filament formation through core-driven polymerization and lateral associations through CTP-mediated interactions. We also find that the CTL weakens GTP binding while enhancing the catalytic rate, whereas the CTP has opposite effects. The joint contributions of the CTL and CTP make Bs-FtsZ, an enzyme that is only half as efficient as a truncated version that lacks the CTT. Overall, our data suggest that the CTT acts as an auto-regulator of Bs-FtsZ assembly and as an auto-inhibitor of enzymatic activity. Based on our results, we propose hypotheses regarding the hypervariability of CTLs and compare FtsZs to other bacterial proteins with tethered IDRs.

Information theoretic measures for quantifying sequence-ensemble relationships of intrinsically disordered proteins.

Intrinsically disordered proteins (IDPs) contribute to a multitude of functions. De novo design of IDPs should open the door to modulating functions and phenotypes controlled by these systems. Recent design efforts have focused on compositional biases and specific sequence patterns as the design features. Analysis of the impact of these designs on sequence-function relationships indicates that individual sequence/compositional parameters are insufficient for describing sequence-function relationships in IDPs. To remedy this problem, we have developed information theoretic measures for sequence-ensemble relationships (SERs) of IDPs. These measures rely on prior availability of statistically robust conformational ensembles derived from all atom simulations. We show that the measures we have developed are useful for comparing sequence-ensemble relationships even when sequence is poorly conserved. Based on our results, we propose that de novo designs of IDPs, guided by knowledge of their SERs, should provide improved insights into their sequence-ensemble-function relationships.

Sequence-to-Conformation Relationships of Disordered Regions Tethered to Folded Domains of Proteins.

Intrinsically disordered proteins and regions (IDPs/IDRs) are characterized by well-defined sequence-to-conformation relationships (SCRs). These relationships refer to the sequence-specific preferences for average sizes, shapes, residue-specific secondary structure propensities, and amplitudes of multiscale conformational fluctuations. SCRs are discerned from the sequence-specific conformational ensembles of IDPs. A vast majority of IDPs are actually tethered to folded domains (FDs). This raises the question of whether or not SCRs inferred for IDPs are applicable to IDRs tethered to FDs. Here, we use atomistic simulations based on a well-established forcefield paradigm and an enhanced sampling method to obtain comparative assessments of SCRs for 13 archetypal IDRs modeled as autonomous units, as C-terminal tails connected to FDs, and as linkers between pairs of FDs. Our studies uncover a set of general observations regarding context-independent versus context-dependent SCRs of IDRs. SCRs are minimally perturbed upon tethering to FDs if the IDRs are deficient in charged residues and for polyampholytic IDRs where the oppositely charged residues within the sequence of the IDR are separated into distinct blocks. In contrast, the interplay between IDRs and tethered FDs has a significant modulatory effect on SCRs if the IDRs have intermediate fractions of charged residues or if they have sequence-intrinsic conformational preferences for canonical random coils. Our findings suggest that IDRs with context-independent SCRs might be independent evolutionary modules, whereas IDRs with context-dependent SCRs might co-evolve with the FDs to which they are tethered.

Regulation of human nucleus pulposus cells by peptide-coupled substrates.

Nucleus pulposus (NP) cells are derived from the notochord and differ from neighboring cells of the intervertebral disc in phenotypic marker expression and morphology. Adult human NP cells lose this phenotype and morphology with age in a pattern that contributes to progressive disc degeneration and pathology. Select laminin-mimetic peptide ligands and substrate stiffnesses were examined for their ability to regulate human NP cell phenotype and biosynthesis through the expression of NP-specific markers aggrecan, N-cadherin, collagen types I and II, and GLUT1. Peptide-conjugated substrates demonstrated an ability to promote expression of healthy NP-specific markers, as well as increased biosynthetic activity. We show an ability to re-express markers of the juvenile NP cell and morphology through control of peptide presentation and stiffness on well-characterized polyacrylamide substrates. NP cells cultured on surfaces conjugated with α3 integrin receptor peptides P4 and P678, and on α2, α5, α6, ß1 integrin-recognizing peptide AG10, show increased expression of aggrecan, N-cadherin, and types I and II collagen, suggesting a healthier, more juvenile-like phenotype. Multi-cell cluster formation was also observed to be more prominent on peptide-conjugated substrates. These findings indicate a critical role for cell-matrix interactions with specific ECM-mimetic peptides in supporting and maintaining a healthy NP cell phenotype and bioactivity. STATEMENT OF SIGNIFICANCE: NP cells reside in a laminin-rich environment that deteriorates with age, including a loss of water content and changes in the extracellular matrix (ECM) structure that may lead to the development of a degenerated IVD. There is great interest in methods to re-express healthy, biosynthetically active NP cells using laminin-derived biomimetic peptides toward the goal of using autologous cell sources for tissue regeneration. Here, we describe a novel study utilizing several laminin mimetic peptides conjugated to polyacrylamide gels that are able to support an immature, healthy NP phenotype after culture on "soft" peptide gels. These findings can support future studies in tissue regeneration where cells may be directed to a desired regenerative phenotype using niche-specific ECM peptides.