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Among the 67 predicted TonB-dependent outer membrane transporters of Caulobacter crescentus, NagA was found to be essential for growth on N-acetyl-␤-D-glucosamine (GlcNAc) and larger chitin oligosaccharides. NagA (93 kDa) has a predicted typical domain structure of an outer membrane transport protein: a signal sequence, the TonB box EQVVIT, a hatch domain of 147 residues, and a ␤-barrel composed of 22 antiparallel ␤-strands linked by large surface loops and very short periplasmic turns. Mutations in tonB1 and exbBD, known to be required for maltose transport via MalA in C. crescentus, and in two additional predicted tonB genes (open reading frames cc2327 and cc3508) did not affect NagA-mediated GlcNAc uptake. nagA is located in a gene cluster that encodes a predicted PTS sugar transport system and two enzymes that convert GlcNAc-6-P to fructose-6-P. Since a nagA insertion mutant did not grow on and transport GlcNAc, diffusion of GlcNAc through unspecific porins in the outer membrane is excluded. Uptake of GlcNAc into tonB and exbBD mutants and reduction but not abolishment of GlcNAc transport by agents which dissipate the electrochemical potential of the cytoplasmic membrane (0.1 mM carbonyl cyanide 3-chlorophenylhydrazone and 1 mM 2,4-dinitrophenol) suggest diffusion of GlcNAc through a permanently open pore of NagA. Growth on (GlcNAc) 3 and (GlcNAc) 5 requires ExbB and ExbD, indicating energy-coupled transport by NagA. We propose that NagA forms a small pore through which GlcNAc specifically diffuses into the periplasm and functions as an energy-coupled transporter for the larger chitin oligosaccharides.TonB-dependent outer membrane proteins actively transport Fe 3ϩ siderophores, heme, Fe 3ϩ , and vitamin B 12 across the outer membrane of gram-negative bacteria (4,6,39,40,52,54). It is thought that energy is required for two steps: (i) release of the tightly bound substrates from the transporters and (ii) movement of the hatch to open a pore in the ␤-barrel (10, 53). The energy is provided by the proton motive force of the cytoplasmic membrane. A protein complex composed of TonB, ExbB, and ExbD is involved in the energy transfer from the cytoplasmic membrane to the outer membrane. TonB interacts with the TonB box in the N-terminal region of the transporters (9, 36, 37, 48). In Escherichia coli, eight such transporters have been related to the transport of specific substrates.Analysis of the genome sequence of Caulobacter crescentus predicts 67 TonB-dependent outer membrane proteins (32, 33). We have identified the tonB, exbB, and exbD genes, required for the transport of maltose and larger maltodextrins (29, 32). Transport is mediated by the outer membrane protein MalA (32). malA, tonB, exbB, and exbD gene insertion mutants transport maltose at 2% of the wild-type rate. This was the first description of TonB-dependent energy-coupled transport across the outer membrane for substrates other than Fe 3ϩ , Fe 3ϩ chelates, and vitamin B 12 .In nature, maltodextrins are derived from abundant starch. It is reasonable ...

Combinatorial assembly of protein domains plays an important role in the evolution of proteins. There is also evidence that protein domains have come together from stable subdomains. This concept of modular assembly could be used to construct new well folded proteins from stable protein fragments. Here, we report the construction of a chimeric protein from parts of a (␤␣)8-barrel enzyme from histidine biosynthesis pathway (HisF) and a protein of the (␤␣)5-flavodoxin-like fold (CheY) from Thermotoga maritima that share a high structural similarity. We expected this construct to fold into a full (␤␣)8-barrel. Our results show that the chimeric protein is a stable monomer that unfolds with high cooperativity. Its three-dimensional structure, which was solved to 3.1 Å resolution by x-ray crystallography, confirms a barrel-like fold in which the overall structures of the parent proteins are highly conserved. The structure further reveals a ninth strand in the barrel, which is formed by residues from the HisF C terminus and an attached tag. This strand invades between ␤-strand 1 and 2 of the CheY part closing a gap in the structure that might be due to a suboptimal fit between the fragments. Thus, by a combination of parts from two different folds and a small arbitrary fragment, we created a well folded and stable protein.chimeric protein ͉ enzyme evolution ͉ flavodoxin-like fold ͉ protein design ͉ TIM-barrel

It is hypothesized that protein domains evolved from smaller intrinsically stable subunits via combinatorial assembly. Illegitimate recombination of fragments that encode protein subunits could have quickly led to diversification of protein folds and their functionality. This evolutionary concept presents an attractive strategy to protein engineering, e.g., to create new scaffolds for enzyme design. We previously combined structurally similar parts from two ancient protein folds, the (βα) 8 -barrel and the flavodoxin-like fold. The resulting "hopeful monster" differed significantly from the intended (βα) 8 -barrel fold by an extra β-strand in the core. In this study, we ask what modifications are necessary to form the intended structure and what potential this approach has for the rational design of functional proteins. Guided by computational design, we optimized the interface between the fragments with five targeted mutations yielding a stable, monomeric protein whose predicted structure was verified experimentally. We further tested binding of a phosphorylated compound and detected that some affinity was already present due to an intact phosphate-binding site provided by one fragment. The affinity could be improved quickly to the level of natural proteins by introducing two additional mutations. The study illustrates the potential of recombining protein fragments with unique properties to design new and functional proteins, offering both a possible pathway of protein evolution and a protocol to rapidly engineer proteins for new applications. T oday's protein world is extremely diverse. It evolved to facilitate a large variety of functions. However, careful analysis revealed that many proteins of different folds share fragments that are structurally similar. 1 This observation led to the proposition that protein domains evolved by combinatorial assembly of smaller gene fragments that encode intrinsically stable subunits. 2,3 Illegitimate recombination of such subunits could have quickly led to diversification of domain architecture, generating proteins from which new folds and functions could have emerged. Here, we present compelling experimental evidence for this hypothesis by demonstrating that fragments from contemporary proteins are easily adapted to form a new protein with selectable properties (Figure 1). Furthermore, this successful rational design is proof of principle that fragment recruitment from present-day proteins can be used to generate new scaffolds with ready-made and easily adaptable properties.Recent successful approaches in computational enzyme design construct a new catalytic site into known protein scaffolds. 4,5 Thus, it would be advantageous to start with a protein that already has the propensity for a certain type of reaction, analogous to how evolution recruits protein scaffolds, or fragments thereof, that then evolve into specialized enzymes.For the present study, protein fragments from two major folds were selected: the TIM-or (βα) 8 -barrel and the flavodoxin-like fold. ...

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