Christian Cambillau

Recognition of antigens by single-domain antibody fragments: the superfluous luxury of paired domains

The antigen-binding site of antibodies from vertebrates is formed by combining the variable domains of a heavy chain (VH) and a light chain (VL). However, antibodies from camels and llamas are an important exception to this in that their sera contain, in addition, a unique kind of antibody that is formed by heavy chains only. The antigen-binding site of these antibodies consists of one single domain, referred to as VHH. This article reviews the mutations and structural adaptations that have taken place to reshape a VH of a VH-VL pair into a single-domain VHH with retention of a sufficient variability. The VHH has a potent antigen-binding capacity and provides the advantage of interacting with novel epitopes that are inaccessible to conventional VH-VL pairs.

Crystal structures of the bovine beta4galactosyltransferase catalytic domain and its complex with uridine diphosphogalactose.

β1,4‐galactosyltransferase T1 (β4Gal‐T1, EC 2.4.1.90/38), a Golgi resident membrane‐bound enzyme, transfers galactose from uridine diphosphogalactose to the terminal β‐N‐acetylglucosamine residues forming the poly‐N‐acetyllactosamine core structures present in glycoproteins and glycosphingolipids. In mammals, β4Gal‐T1 binds to α‐lactalbumin, a protein that is structurally homologous to lyzozyme, to produce lactose. β4Gal‐T1 is a member of a large family of homologous β4galactosyltransferases that use different types of glycoproteins and glycolipids as substrates. Here we solved and refined the crystal structures of recombinant bovine β4Gal‐T1 to 2.4 Å resolution in the presence and absence of the substrate uridine diphosphogalactose. The crystal structure of the bovine substrate‐free β4Gal‐T1 catalytic domain showed a new fold consisting of a single conical domain with a large open pocket at its base. In the substrate‐bound complex, the pocket encompassed residues interacting with uridine diphosphogalactose. The structure of the complex contained clear regions of electron density for the uridine diphosphate portion of the substrate, where its β‐phosphate group was stabilized by hydrogen‐bonding contacts with conserved residues including the Asp252ValAsp254 motif. These results help the interpretation of engineered β4Gal‐T1 point mutations. They suggest a mechanism possibly involved in galactose transfer and enable identification of the critical amino acids involved in α‐lactalbumin interactions.

Structural and Genomic Correlates of Hyperthermostability

While most organisms grow at temperatures ranging between 20 and 50 °C, many archaea and a few bacteria have been found capable of withstanding temperatures close to 100 °C, or beyond, such as Pyrococcus or Aquifex. Here we report the results of two independent large scale unbiased approaches to identify global protein properties correlating with an extreme thermophile lifestyle. First, we performed a comparative proteome analyses using 30 complete genome sequences from the three kingdoms. A large difference between the proportions of charged versuspolar (noncharged) amino acids was found to be a signature of all hyperthermophilic organisms. Second, we analyzed the water accessible surfaces of 189 protein structures belonging to mesophiles or hyperthermophiles. We found that the surfaces of hyperthermophilic proteins exhibited the shift already observed at the genomic level,i.e. a proportion of solvent accessible charged residues strongly increased at the expense of polar residues. The biophysical requirements for the presence of charged residues at the protein surface, allowing protein stabilization through ion bonds, is therefore clearly imprinted and detectable in all genome sequences available to date.

A novel type of catalytic copper cluster in nitrous oxide reductase

Nitrous oxide (N2O) is a greenhouse gas, the third most significant contributor to global warming. As a key process for N2O elimination from the biosphere, N2O reductases catalyze the two-electron reduction of N2O to N2. These 2 × 65 kDa copper enzymes are thought to contain a CuA electron entry site, similar to that of cytochrome c oxidase, and a CuZ catalytic center. The copper anomalous signal was used to solve the crystal structure of N2O reductase from Pseudomonas nautica by multiwavelength anomalous dispersion, to a resolution of 2.4 Å. The structure reveals that the CuZ center belongs to a new type of metal cluster, in which four copper ions are liganded by seven histidine residues. N2O binds to this center via a single copper ion. The remaining copper ions might act as an electron reservoir, assuring a fast electron transfer and avoiding the formation of dead-end products.

Conformational and functional similarities between glutaredoxin and thioredoxins.

The tertiary structures of thioredoxin from Escherichia coli and bacteriophage T4 have been compared and aligned giving a common fold of 68 C alpha atoms with a root mean square difference of 2.6 A. The amino acid sequence of glutaredoxin has been aligned to those of the thioredoxins assuming that glutaredoxin has the same common fold. A model of the glutaredoxin molecule was built on a vector display using this alignment and the T4 thioredoxin tertiary structure. By comparison of the model with those of the thioredoxins, we have identified a molecular surface area on one side of the redox‐active S‐S bridge which we suggest is the binding area of these molecules for redox interactions with other proteins. This area comprises residues 33‐34, 75‐76 and 91‐93 in E. coli thioredoxin; 15‐16, 65‐66 and 76‐78 in T4 thioredoxin and 12‐13, 59‐60 and 69‐71 in glutaredoxin. In all three molecules, this part of the surface is flat and hydrophobic. Charged groups are completely absent. In contrast, there is a cluster of charged groups on the other side of the S‐S bridge which we suggest participates in the mechanisms of the redox reactions. In particular, a lysine residue close to an aromatic ring is conserved in all molecules.

A Common Evolutionary Origin for Tailed-Bacteriophage Functional Modules and Bacterial Machineries

SUMMARY Bacteriophages belonging to the order Caudovirales possess a tail acting as a molecular nanomachine used during infection to recognize the host cell wall, attach to it, pierce it, and ensure the high-efficiency delivery of the genomic DNA to the host cytoplasm. In this review, we provide a comprehensive analysis of the various proteins constituting tailed bacteriophages from a structural viewpoint. To this end, we had in mind to pinpoint the resemblances within and between functional modules such as capsid/tail connectors, the tails themselves, or the tail distal host recognition devices, termed baseplates. This comparison has been extended to bacterial machineries embedded in the cell wall, for which shared molecular homology with phages has been recently revealed. This is the case for the type VI secretion system (T6SS), an inverted phage tail at the bacterial surface, or bacteriocins. Gathering all these data, we propose that a unique ancestral protein fold may have given rise to a large number of bacteriophage modules as well as to some related bacterial machinery components.

Crystal structure of the bifunctional N-acetylglucosamine 1-phosphate uridyltransferase from Escherichia coli: a paradigm for the related pyrophosphorylase superfamily

N‐acetylglucosamine 1‐phosphate uridyltransferase (GlmU) is a cytoplasmic bifunctional enzyme involved in the biosynthesis of the nucleotide‐activated UDP‐GlcNAc, which is an essential precursor for the biosynthetic pathways of peptidoglycan and other components in bacteria. The crystal structure of a truncated form of GlmU has been solved at 2.25 Å resolution using the multiwavelength anomalous dispersion technique and its function tested with mutagenesis studies. The molecule is composed of two distinct domains connected by a long α‐helical arm: (i) an N‐terminal domain which resembles the dinucleotide‐binding Rossmann fold; and (ii) a C‐terminal domain which adopts a left‐handed parallel β‐helix structure (LβH) as found in homologous bacterial acetyltransferases. Three GlmU molecules assemble into a trimeric arrangement with tightly packed parallel LβH domains, the long α‐helical linkers being seated on top of the arrangement and the N‐terminal domains projected away from the 3‐fold axis. In addition, the 2.3 Å resolution structure of the GlmU–UDP‐GlcNAc complex reveals the structural bases required for the uridyltransferase activity. These structures exemplify a three‐dimensional template for the development of new antibacterial agents and for studying other members of the large family of XDP‐sugar bacterial pyrophosphorylases.

High-throughput automated refolding screening of inclusion bodies

One of the main stumbling blocks encountered when attempting to express foreign proteins in Escherichia coli is the occurrence of amorphous aggregates of misfolded proteins, called inclusion bodies (IB). Developing efficient protein native structure recovery procedures based on IB refolding is therefore an important challenge. Unfortunately, there is no “universal” refolding buffer: Experience shows that refolding buffer composition varies from one protein to another. In addition, the methods developed so far for finding a suitable refolding buffer suffer from a number of weaknesses. These include the small number of refolding formulations, which often leads to negative results, solubility assays incompatible with high‐throughput, and experiment formatting not suitable for automation. To overcome these problems, it was proposed in the present study to address some of these limitations. This resulted in the first completely automated IB refolding screening procedure to be developed using a 96‐well format. The 96 refolding buffers were obtained using a fractional factorial approach. The screening procedure is potentially applicable to any nonmembrane protein, and was validated with 24 proteins in the framework of two Structural Genomics projects. The tests used for this purpose included the use of quality control methods such as circular dichroism, dynamic light scattering, and crystallogenesis. Out of the 24 proteins, 17 remained soluble in at least one of the 96 refolding buffers, 15 passed large‐scale purification tests, and five gave crystals.

Structural biology of type VI secretion systems

Type VI secretion systems (T6SSs) are transenvelope complexes specialized in the transport of proteins or domains directly into target cells. These systems are versatile as they can target either eukaryotic host cells and therefore modulate the bacteria–host interaction and pathogenesis or bacterial cells and therefore facilitate access to a specific niche. These molecular machines comprise at least 13 proteins. Although recent years have witnessed advances in the role and function of these secretion systems, little is known about how these complexes assemble in the cell envelope. Interestingly, the current information converges to the idea that T6SSs are composed of two subassemblies, one resembling the contractile bacteriophage tail, whereas the other subunits are embedded in the inner and outer membranes and anchor the bacteriophage-like structure to the cell envelope. In this review, we summarize recent structural information on individual T6SS components emphasizing the fact that T6SSs are composite systems, adapting subunits from various origins.

Structural basis for the substrate selectivity of pancreatic lipases and some related proteins

The classical human pancreatic lipase (HPL), the guinea pig pancreatic lipase-related protein 2 (GPLRP2) and the phospholipase A1 from hornet venom (DolmI PLA1) illustrate three interesting steps in the molecular evolution of the pancreatic lipase gene family towards different substrate selectivities. Based on the known 3D structures of HPL and a GPLRP2 chimera, as well as the modeling of DolmI PLA1, we review here the structural features and the kinetic properties of these three enzymes for a better understanding of their structure-function relationships. HPL displays significant activity only on triglycerides, whereas GPLRP2 displays high phospholipase and galactolipase activities, together with a comparable lipase activity. GPLRP2 shows high structural homology with HPL with the exception of the lid domain which is made of five amino acid residues (mini-lid) instead of 23 in HPL. The lid domain deletion in GPLRP2 allows the free access to the active site and reduces the steric hindrance towards large substrates, such as galactolipids. The role of the lid domain in substrate selectivity has been investigated by site-directed mutagenesis and the substitution of HPL and GPLRP2 lid domains. The addition of a large-size lid domain in GPLRP2 increases the substrate selectivity for triglycerides by depressing the phospholipase activity. The phospholipase activity is, however, not induced in the case of the HPL mutant with GPLRP2 mini-lid. Therefore, the presence of a full-length lid domain is not the unique structural feature explaining the absence of phospholipase activity in HPL. The 3D structure of the GPLRP2 chimera and the model of DolmI PLA1 reveal a higher hydrophilic/lipophilic balance (HLB) of the surface loops (beta5 loop, beta9 loop, lid domain) surrounding the active site, as compared to the homologous loops in HPL. This observation provides a potential explanation for the ability of GPLRP2 and DolmI PLA1 to hydrolyze polar lipids, such as phospholipids. In conclusion, the beta5 loop, the beta9 loop, and the lid domain play an essential role in substrate selectivity towards triglycerides, phospholipids and galactolipids.