Pneumococcal bacteriophage-encoded lysins are modular choline binding proteins that have been shown to act as enzymatic antimicrobial agents (enzybiotics) against streptococcal infections. Here we present the crystal structures of the free and choline bound states of the Cpl-1 lysin, encoded by the pneumococcal phage Cp-1. While the catalytic module displays an irregular (beta/alpha)(5)beta(3) barrel, the cell wall-anchoring module is formed by six similar choline binding repeats (ChBrs), arranged into two different structural regions: a left-handed superhelical domain configuring two choline binding sites, and a beta sheet domain that contributes in bringing together the whole structure. Crystallographic and site-directed mutagenesis studies allow us to propose a general catalytic mechanism for the whole glycoside hydrolase family 25. Our work provides the first complete structure of a member of the large family of choline binding proteins and reveals that ChBrs are versatile elements able to tune the evolution and specificity of the pneumococcal surface proteins.

Pmel17 is a melanocyte protein necessary for eumelanin deposition 1 in mammals and found in melanosomes in a filamentous form. The luminal part of human Pmel17 includes a region (RPT) with 10 copies of a partial repeat sequence, pt.e.gttp.qv., known to be essential in vivo for filament formation. We show that this RPT region readily forms amyloid in vitro, but only under the mildly acidic conditions typical of the lysosome-like melanosome lumen, and the filaments quickly become soluble at neutral pH. Under the same mildly acidic conditions, the Pmel filaments promote eumelanin formation. Electron diffraction, circular dichroism, and solidstate NMR studies of Pmel17 filaments show that the structure is rich in beta sheet. We suggest that RPT is the amyloid core domain of the Pmel17 filaments so critical for melanin formation.

Macromolecular condensation resulting from biologically regulated liquid–liquid phase separation is emerging as a mechanism to organize intracellular space in eukaryotes, with broad implications for cell physiology and pathology. Despite their small size, bacterial cells are also organized by proteins such as FtsZ, a tubulin homolog that assembles into a ring structure precisely at the cell midpoint and is required for cytokinesis. Here, we demonstrate that FtsZ can form crowding‐induced condensates, reminiscent of those observed for eukaryotic proteins. Formation of these FtsZ‐rich droplets occurs when FtsZ is bound to SlmA, a spatial regulator of FtsZ that antagonizes polymerization, while also binding to specific sites on chromosomal DNA. The resulting condensates are dynamic, allowing FtsZ to undergo GTP‐driven assembly to form protein fibers. They are sensitive to compartmentalization and to the presence of a membrane boundary in cell mimetic systems. This is a novel example of a bacterial nucleoprotein complex exhibiting condensation into liquid droplets, suggesting that phase separation may also play a functional role in the spatiotemporal organization of essential bacterial processes.

The assembly of the bacterial cell division FtsZ protein in the presence of constantly replenished GTP was studied as a function of Mg2+ concentration (at neutral pH and 0.5 M potassium) under steady-state conditions by sedimentation velocity, concentration-gradient light scattering, fluorescence correlation spectroscopy and dynamic light scattering. Sedimentation velocity measurements confirmed previous results indicating cooperative appearance of a narrow size distribution of finite oligomers with increasing protein concentration. The concentration dependence of light scattering and diffusion coefficients independently verified the cooperative appearance of a narrow distribution of high molecular weight oligomers, and in addition provided a measurement of the average size of these species, which corresponds to 100 ± 20 FtsZ protomers at millimolar Mg2+ concentration. Parallel experiments on solutions containing GMPCPP, a slowly hydrolysable analog of GTP, in place of GTP, likewise indicated the concerted formation of a narrow size distribution of fibrillar oligomers with a larger average mass (corresponding to 160 ± 20 FtsZ monomers). The closely similar behavior of FtsZ in the presence of both GTP and GMPCPP suggests that the observations reflect equilibrium rather than non-equilibrium steady-state properties of both solutions and exhibit parallel manifestations of a common association scheme.

The influence of membrane-free microcompartments resulting from crowding-induced liquid/liquid phase separation (LLPS) on the dynamic spatial organization of FtsZ, the main component of the bacterial division machinery, has been studied using several LLPS systems. The GTP-dependent assembly cycle of FtsZ is thought to be crucial for the formation of the septal ring, which is highly regulated in time and space. We found that FtsZ accumulates in one of the phases and/or at the interface, depending on the system composition and on the oligomerization state of the protein. These results were observed both in bulk LLPS and in lipid-stabilized, phase-separated aqueous microdroplets. The visualization of the droplets revealed that both the location and structural arrangement of FtsZ filaments is determined by the nature of the LLPS. Relocation upon depolymerization of the dynamic filaments suggests the protein may shift among microenvironments in response to changes in its association state. The existence of these dynamic compartments driven by phase transitions can alter the local composition and reactivity of FtsZ during its life cycle acting as a nonspecific modulating factor of cell function.

Pneumococcal bacteriophage-encoded lysins are modular proteins that have been shown to act as enzymatic antimicrobial agents (enzybiotics) in treatment of streptococcal infections. The first x-ray crystal structures of the Cpl-1 lysin, encoded by the pneumococcal phage Cp-1, in complex with three bacterial cell wall peptidoglycan (PG) analogues are reported herein. The Cpl-1 structure is folded in two well defined modules, one responsible for anchoring to the pneumococcal cell wall and the other, a catalytic module, that hydrolyzes the PG. Conformational rearrangement of Tyr-127 is a critical event in molecular recognition of a stretch of five saccharide rings of the polymeric peptidoglycan (cell wall). The PG is bound at a stretch of the surface that is defined as the peptidoglycan-binding sites 1 and 2, the juncture of which catalysis takes place. The peptidoglycan-binding site 1 binds to a stretch of three saccharides of the peptidoglycan in a conformation essentially identical to that of the peptidoglycan in solution. In contrast, binding of two peptidoglycan saccharides at the peptidoglycan-binding site 2 introduces a kink into the solution structure of the peptidoglycan, en route to catalytic turnover. These findings provide the first structural evidence on recognition of the peptidoglycan and shed light on the discrete events of cell wall degradation by Cpl-1.Streptococcus pneumoniae is one of the most common and important human pathogens, which causes serious life-threatening diseases such as acute otitis media, pneumonia, sepsis, and meningitis. Pneumococcal infections are associated with high morbidity and mortality, especially among children, the elderly, and the immune-depressed patients. The widespread emergence of antibiotic resistance and the lack of highly effective pneumococcal vaccines against all serotypes of this organism give urgency to elucidation of the molecular processes involved in its pathogenicity (1, 2).The peptidoglycan (PG) 3 scaffold of the bacterial cell wall is a repeating GlcNAc-N-acetylmuramic (MurNAc) disaccharide (GlcNAc-(␤-1,4)-MurNAc) unit having a pentapeptide attached to the D-lactyl moiety of each MurNAc unit. All known pneumococcal bacteriophages encode an amidase or a lysozyme, which hydrolyzes the PG at the final stage of the phage reproductive cycle, leading to bacterial cell lysis. These enzymes, known collectively as endolysins, have been shown to be highly efficient in killing pneumococci in vitro and can eradicate this organism from the upper respiratory tract or from the bloodstream of mice (3, 4) acting as new antimicrobial agents (i.e. enzybiotics). In addition, Cpl-1 lysin and Pal amidase encoded by phage Dp-1 act in a synergistic manner in a sepsis mouse model (5); this synergy has also been confirmed in in vitro experiments with Cpl-1 and penicillin or gentamicin (6). Very recently, the creation of a new animal model of otitis media has been reported (7). Using this new mouse model, it has been demonstrated that Cpl-1 could eliminate colonization with S. p...

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Pal amidase, encoded by pneumococcal bacteriophage Dp-1, represents one step beyond in the modular evolution of pneumococcal murein hydrolases. It exhibits the choline-binding module attaching pneumococcal lysins to the cell wall, but the catalytic module is different from those present in the amidases coded by the host or other pneumococcal phages. Pal is also an effective antimicrobial agent against Streptococcus pneumoniae that may constitute an alternative to antibiotic prophylaxis. The structural implications of Pal singular structure and their effect on the choline-amidase interactions have been examined by means of several techniques. Pal stability is maximum around pH 8.0 (T m Х 50.2°C; ⌬H t ‫؍‬ 183 ؎ 4 kcal mol ؊1 ), and its constituting modules fold as two tight interacting cooperative units whose denaturation merges into a single process in the free amidase but may proceed as two well resolved events in the choline-bound state. Choline titration curves reflect low energy ligand-protein interactions and are compatible with two sets of sites. Choline binding strongly stabilizes the cell wall binding module, and the conformational stabilization is transmitted to the catalytic region. Moreover, the high proportion of aggregates formed by the unbound amidase together with choline preferential interaction with Pal dimers suggest the existence of marginally stable regions that would become stabilized through choline-protein interactions without significantly modifying Pal secondary structure. This structural rearrangement may underlie in vitro "conversion" of Pal from the low to the full activity form triggered by choline. The Pal catalytic module secondary structure could denote folding conservation within pneumococcal lytic amidases, but the number of functional choline binding sites is reduced (2-3 sites per monomer) when compared with pneumococcal LytA amidase (4 -5 sites per monomer) and displays different intermodular interactions.Dp-1, the first described pneumococcal bacteriophage, belongs to the Siphoviridae family (1). Its peptidoglycan-degrading enzyme, Pal, 1 was biochemically characterized as a cholinedependent amidase (2) synthesized as a low activity form that requires in vitro incubation with choline or choline-containing cell walls to achieve full activity (3), a process designated as "conversion." Pal shows the modular organization characteristic of pneumococcal cell wall lysins (3), but represents one step beyond in the modular evolution of pneumococcal murein hydrolases. Its N-terminal region has no similarity with the amidases coded by the host or other pneumococcal phages (4, 5), 2 but is highly similar to the murein hydrolase coded by Lactococcus phage BK5-T. 3 The C-terminal region comprises a choline binding module (ChBM) homologous to that found in the pneumococcal lytic system 4 (6) that attaches the enzyme to choline residues present in pneumococcal envelope (7). As in most pneumococcal lysins so far known, six repeats of about 20 amino acids (p1-p6) and a short C-terminal tail fo...