Ethan C. Garner

Sequence complexity of disordered protein

Intrinsic disorder refers to segments or to whole proteins that fail to self‐fold into fixed 3D structure, with such disorder sometimes existing in the native state. Here we report data on the relationships among intrinsic disorder, sequence complexity as measured by Shannons entropy, and amino acid composition. Intrinsic disorder identified in protein crystal structures, and by nuclear magnetic resonance, circular dichroism, and prediction from amino acid sequence, all exhibit similar complexity distributions that are shifted to lower values compared to, but significantly overlapping with, the distribution for ordered proteins. Compared to sequences from ordered proteins, these variously characterized intrinsically disordered segments and proteins, and also a collection of low‐complexity sequences, typically have obviously higher levels of protein‐specific subsets of the following amino acids: R, K, E, P, and S, and lower levels of subsets of the following: C, W, Y, I, and V. The Swiss Protein database of sequences exhibits significantly higher amounts of both low‐complexity and predicted‐to‐be‐disordered segments as compared to a non‐redundant set of sequences from the Protein Data Bank, providing additional data that nature is richer in disordered and low‐complexity segments compared to the commonness of these features in the set of structurally characterized proteins. Proteins 2001;42:38–48.

Coupled, Circumferential Motions of the Cell Wall Synthesis Machinery and MreB Filaments in B. subtilis

Bacteria elongation involves moving synthetic complexes around the cell wall. Rod-shaped bacteria elongate by the action of cell wall synthesis complexes linked to underlying dynamic MreB filaments. To understand how the movements of these filaments relate to cell wall synthesis, we characterized the dynamics of MreB and the cell wall elongation machinery using high-precision particle tracking in Bacillus subtilis. We found that MreB and the elongation machinery moved circumferentially around the cell, perpendicular to its length, with nearby synthesis complexes and MreB filaments moving independently in both directions. Inhibition of cell wall synthesis by various methods blocked the movement of MreB. Thus, bacteria elongate by the uncoordinated, circumferential movements of synthetic complexes that insert radial hoops of new peptidoglycan during their transit, possibly driving the motion of the underlying MreB filaments.

A spindle-like apparatus guides bacterial chromosome segregation

Until recently, a dedicated mitotic apparatus that segregates newly replicated chromosomes into daughter cells was believed to be unique to eukaryotic cells. Here we demonstrate that the bacterium Caulobacter crescentus segregates its chromosome using a partitioning (Par) apparatus that has surprising similarities to eukaryotic spindles. We show that the C. crescentus ATPase ParA forms linear polymers in vitro and assembles into a narrow linear structure in vivo. The centromere-binding protein ParB binds to and destabilizes ParA structures in vitro. We propose that this ParB-stimulated ParA depolymerization activity moves the centromere to the opposite cell pole through a burnt bridge Brownian ratchet mechanism. Finally, we identify the pole-specific TipN protein as a new component of the Par system that is required to maintain the directionality of DNA transfer towards the new cell pole. Our results elucidate a bacterial chromosome segregation mechanism that features basic operating principles similar to eukaryotic mitotic machines, including a multivalent protein complex at the centromere that stimulates the dynamic disassembly of polymers to move chromosomes into daughter compartments.

Reconstitution of DNA Segregation Driven by Assembly of a Prokaryotic Actin Homolog

Multiple unrelated polymer systems have evolved to partition DNA molecules between daughter cells at division. To better understand polymer-driven DNA segregation, we reconstituted the three-component segregation system of the R1 plasmid from purified components. We found that the ParR/parC complex can construct a simple bipolar spindle by binding the ends of ParM filaments, inhibiting dynamic instability, and acting as a ratchet permitting incorporation of new monomers and riding on the elongating filament ends. Under steady-state conditions, the dynamic instability of unattached ParM filaments provides the energy required to drive DNA segregation.

THE PROTEIN NON-FOLDING PROBLEM: AMINO ACID DETERMINANTS OF INTRINSIC ORDER AND DISORDER

To investigate the determinants of protein order and disorder, three primary and one derivative database of intrinsically disordered proteins were compiled. The segments in each primary database were characterized by one of the following: X-ray crystallography, nuclear magnetic resonance (NMR), or circular dichroism (CD). The derivative database was based on homology. The three primary disordered databases have a combined total of 157 proteins or segments of length.30 with 18,010 residues, while the derivative database contains 572 proteins from 32 families with 52,688 putatively disordered residues. For the four disordered databases, the amino acid compositions were compared with those from a database of ordered structure. Relative to the ordered protein, the intrinsically disordered segments in all four databases were significantly depleted in W, C, F, I, Y, V, L and N, significantly enriched in A, R, G, Q, S, P, E and K, and inconsistently different in H, M, T, and D, suggesting that the first set be called order-promoting and the second set disorder-promoting. Also, 265 amino acid properties were ranked by their ability to discriminate order and disorder and then pruned to remove the most highly correlated pairs. The 10 highest-ranking properties after pruning consisted of 2 residue contact scales, 4 hydrophobicity scales, 3 scales associated with.-sheets and one polarity scale. Using these 10 properties for comparisons of the 3 primary databases suggests that disorder in all 3 databases is very similar, but with those characterized by NMR and CD being the most similar, those by CD and X-ray being next, and those by NMR and X-ray being the least similar.

Treadmilling by FtsZ filaments drives peptidoglycan synthesis and bacterial cell division

Coordinating cell wall synthesis and cell division Most bacteria are protected by peptidoglycan cell walls, which must be remodeled to split the cell. Cell division requires the tubulin homolog FtsZ, a highly conserved cytoskeletal polymer that specifies the future site of division. Bisson-Filho et al. and Yang et al. found that the dynamic treadmilling of FtsZ filaments controls both the location and activity of the associated cell wall synthetic enzymes. This creates discrete sites of cell wall synthesis that circle around the division plane to divide the cell. Science, this issue p. 739, p. 744 Bacterial cytokinesis is controlled by the circumferential treadmilling of FtsZ and FtsA filaments that drives the insertion of new cell wall. The mechanism by which bacteria divide is not well understood. Cell division is mediated by filaments of FtsZ and FtsA (FtsAZ) that recruit septal peptidoglycan-synthesizing enzymes to the division site. To understand how these components coordinate to divide cells, we visualized their movements relative to the dynamics of cell wall synthesis during cytokinesis. We found that the division septum was built at discrete sites that moved around the division plane. FtsAZ filaments treadmilled circumferentially around the division ring and drove the motions of the peptidoglycan-synthesizing enzymes. The FtsZ treadmilling rate controlled both the rate of peptidoglycan synthesis and cell division. Thus, FtsZ treadmilling guides the progressive insertion of new cell wall by building increasingly smaller concentric rings of peptidoglycan to divide the cell.

Identification of intrinsic order and disorder in the DNA repair protein XPA

The DNA‐repair protein XPA is required to recognize a wide variety of bulky lesions during nucleotide excision repair. Independent NMR solution structures of a human XPA fragment comprising approximately 40% of the full‐length protein, the minimal DNA‐binding domain, revealed that one‐third of this molecule was disordered. To better characterize structural features of full‐length XPA, we performed time‐resolved trypsin proteolysis on active recombinant Xenopus XPA (xXPA). The resulting proteolytic fragments were analyzed by electrospray ionization interface coupled to a Fourier transform ion cyclotron resonance mass spectrometry and SDS‐PAGE. The molecular weight of the full‐length xXPA determined by mass spectrometry (30922.02 daltons) was consistent with that calculated from the sequence (30922.45 daltons). Moreover, the mass spectrometric data allowed the assignment of multiple xXPA fragments not resolvable by SDS‐PAGE. The neural network program Predictor of Natural Disordered Regions (PONDR) applied to xXPA predicted extended disordered N‐ and C‐terminal regions with an ordered internal core. This prediction agreed with our partial proteolysis results, thereby indicating that disorder in XPA shares sequence features with other well‐characterized intrinsically unstructured proteins. Trypsin cleavages at 30 of the possible 48 sites were detected and no cleavage was observed in an internal region (Q85‐I179) despite 14 possible cut sites. For the full‐length xXPA, there was strong agreement among PONDR, partial proteolysis data, and the NMR structure for the corresponding XPA fragment.

The structure of bacterial ParM filaments

Bacterial ParM is a homolog of eukaryotic actin and is involved in moving plasmids so that they segregate properly during cell division. Using cryo-EM and three-dimensional reconstruction, we show that ParM filaments have a different structure from F-actin, with very different subunit-subunit interfaces. These interfaces result in the helical handedness of the ParM filament being opposite to that of F-actin. Like F-actin, ParM filaments have a variable twist, and we show that this involves domain-domain rotations within the ParM subunit. The present results yield new insights into polymorphisms within F-actin, as well as the evolution of polymer families.

FtsEX is required for CwlO peptidoglycan hydrolase activity during cell wall elongation in Bacillus subtilis

The peptidoglycan (PG) sacculus, a meshwork of polysaccharide strands cross‐linked by short peptides, protects bacterial cells against osmotic lysis. To enlarge this covalently closed macromolecule, PG hydrolases must break peptide cross‐links in the meshwork to allow insertion of new glycan strands between the existing ones. In the rod‐shaped bacterium Bacillus subtilis, cell wall elongation requires two redundant endopeptidases, CwlO and LytE. However, it is not known how these potentially autolytic enzymes are regulated to prevent lethal breaches in the cell wall. Here, we show that the ATP‐binding cassette transporter‐like FtsEX complex is required for CwlO activity. In Escherichia coli, FtsEX is thought to harness ATP hydrolysis to activate unrelated PG hydrolases during cell division. Consistent with this regulatory scheme, B. subtilis FtsE mutants that are unable to bind or hydrolyse ATP cannot activate CwlO. Finally, we show that in cells depleted of both CwlO and LytE, the PG synthetic machinery continues moving circumferentially until cell lysis, suggesting that cross‐link cleavage is not required for glycan strand polymerization. Overall, our data support a model in which the FtsEX complex is a remarkably flexible regulatory module capable of controlling a diverse set of PG hydrolases during growth and division in different organisms.

Bacterial cell wall biogenesis is mediated by SEDS and PBP polymerase families functioning semi-autonomously

Multi-protein complexes organized by cytoskeletal proteins are essential for cell wall biogenesis in most bacteria. Current models of the wall assembly mechanism assume that class A penicillin-binding proteins (aPBPs), the targets of penicillin-like drugs, function as the primary cell wall polymerases within these machineries. Here, we use an in vivo cell wall polymerase assay in Escherichia coli combined with measurements of the localization dynamics of synthesis proteins to investigate this hypothesis. We find that aPBP activity is not necessary for glycan polymerization by the cell elongation machinery, as is commonly believed. Instead, our results indicate that cell wall synthesis is mediated by two distinct polymerase systems, shape, elongation, division, sporulation (SEDS)-family proteins working within the cytoskeletal machines and aPBP enzymes functioning outside these complexes. These findings thus necessitate a fundamental change in our conception of the cell wall assembly process in bacteria.