During bacterial cell division, the tubulin-homolog FtsZ forms a ring-like structure at the center of the cell. This Z-ring not only organizes the division machinery, but treadmilling of FtsZ filaments was also found to play a key role in distributing proteins at the division site. What regulates the architecture, dynamics and stability of the Z-ring is currently unknown, but FtsZ-associated proteins are known to play an important role. Here, using an in vitro reconstitution approach, we studied how the well-conserved protein ZapA affects FtsZ treadmilling and filament organization into large-scale patterns. Using high-resolution fluorescence microscopy and quantitative image analysis, we found that ZapA cooperatively increases the spatial order of the filament network, but binds only transiently to FtsZ filaments and has no effect on filament length and treadmilling velocity. Together, our data provides a model for how FtsZ-associated proteins can increase the precision and stability of the bacterial cell division machinery in a switch-like manner.

Most bacteria accomplish cell division with the help of a dynamic protein complex called the divisome, which spans the cell envelope in the plane of division. Assembly and activation of this machinery is coordinated by the tubulin-related GTPase FtsZ, which was found to form treadmilling filaments on supported bilayers in vitro 1 and in live cells where they circle around the cell division site 2,3. Treadmilling of FtsZ is thought to actively move proteins around the cell thereby distributing peptidoglycan synthesis and coordinating the inward growth of the septum to form the new poles of the daughter cells 4. However, the molecular mechanisms underlying this function are largely unknown. Here, to study how FtsZ polymerization dynamics are coupled to downstream proteins, we reconstituted part of the bacterial cell division machinery using its purified components FtsZ, FtsA and truncated transmembrane proteins essential for cell division. We found that the membrane-bound cytosolic peptides of FtsN and FtsQ co-migrated with Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:

Background: Structural characterization of integral-membrane (IM) metallopeptidases (MPs) faces enormous technical hurdles. Results:We have discovered a novel family of minimal MPs, minigluzincins, and determined the crystal structures of the zymogens of two family members. Conclusion:Minigluzincins are valid models for catalytic domains of M48 and M56 family IMMPs. Significance: They provide a high resolution scaffold for the design of small molecule inhibitors of IMMPs.

In general, the native fold of a protein in a given environment is unique and at a global free-energy minimum [1] . However, some proteins spontaneously undergo substantial fold switching and reversibly transit between several conformers: "metamorphic" proteins [2] . Unfortunately, identifying and examining such proteins is a challenge because they are highly dynamic and impossible to identify a priori [3] . In contrast, minor rearrangement often occurs in single-domain enzymes upon binding of substrates, as shown for proteolytic enzymes of the metallopeptidase (MP) class [4] . As to enzymatic activity, an increase in enzyme concentration usually raises activity, as more substrate can be bound and turned over [5] . Here we describe a metamorphic minimal selective and specific caseinolytic metallopeptidase, selecase, which shows reversible and concentration-dependent equilibrium between different discrete states and an associated loss of enzymatic activity due to autoinhibition.We recently discovered a family of soluble minimal MPs named "minigluzincins" and characterized two of them, proabylysin and projannalysin, but we only isolated them as inactive zymogens, each in a single conformation [6] . In the present study, we introduce selecase from Methanocaldococcus jannaschii as a novel family member. We recombinantly produced and purified selecase (see Experimental Procedures [EP] and Supplemental Results and Discussion [SRD] in the Supporting Information for details). In contrast to the other minigluzincins, the 110-residue full-length selecase corresponded to a mature, fully active MP with narrow and selective-hitherto unreported-substrate specificity that cleaved bovine milk casein at a single site on its ! s1 chain (Suppl. Fig. 1 and Suppl. Tables 1-2). Selecase was extremely soluble in aqueous buffer and did not precipitate at 130mg/ml. Thus, we studied the concentrationdependent enzymatic activity of selecase on a peptide that mimics the casein cleavage site (peptide CCS). Normally, peptide-bond hydrolysis by MPs is an ordered single-displacement reaction, which follows simple Michaelis-Menten kinetics [7] . This entails that higher enzyme concentrations enhance the initial rate of reaction in the pre-steady state following a hyperbolic curve until a plateau is reached upon saturation [5] . This is found e.g. with tobacco-etch virus proteinase, which is widely used in biotechnology (Fig. 1a). . Tobacco-etch virus proteinase mutant S219V, which shows comparable catalytic efficiency to selecase but normal concentrationdependent activity, is shown for comparison (purple curve). (B) SEC-MALLS of selecase at selected initial concentrations (0.15-65mg/ml; see also Suppl. Fig. 2b). The peak pattern moves towards smaller elution volumes with increasing protein concentration, thus suggesting protein oligomerization. Curves are colored according to the inset in panel (D). (C) Analytical ultracentrifugation curves at six selected concentrations depicting the concentration-dependent oligomeric populations. Es...