Hanna-Kirsti S. Leiros

Cold adapted enzymes

The number of reports on enzymes from cold adapted organisms has increased significantly over the past years, and reveals that adaptive strategies for functioning at low temperature varies among enzymes. However, the high catalytic efficiency at low temperature seems, for the majority of cold active enzymes, to be accompanied by a reduced thermal stability. Increased molecular flexibility to compensate for the low working temperature, is therefore still the most dominating theory for cold adaptation, although there also seem to be other adaptive strategies. The number of experimentally determined 3D structures of enzymes possessing cold adaptation features is still limited, and restricts a structural rationalization for cold activity. The present summary of structural characteristics, based on comparative studies on crystal structures (7), homology models (7), and amino acid sequences (24), reveals that there are no common structural feature that can account for the low stability, increased catalytic efficiency, and proposed molecular flexibility. Analysis of structural features that are thought to be important for stability (e.g. intra-molecular hydrogen bonds and ion-pairs, proline-, methionine-, glycine-, or arginine content, surface hydrophilicity, helix stability, core packing), indicates that each cold adapted enzyme or enzyme system use different small selections of structural adjustments for gaining increased molecular flexibility that in turn give rise to increased catalytic efficiency and reduced stability. Nevertheless, there seem to be a clear correlation between cold adaptation and reduced number of interactions between structural domains or subunits. Cold active enzymes also seem, to a large extent, to increase their catalytic activity by optimizing the electrostatics at and around the active site.

Specific Radiation-Damage Can Be Used To Solve Macromolecular Crystal Structures

The use of third generation synchrotron sources has led to renewed concern about the effect of ionizing radiation on crystalline biological samples. In general, the problem is seen as one to be avoided. However, in this paper, it is shown that, far from being a hindrance to successful structure determination, radiation damage provides an opportunity for phasing macromolecular structures. This is successfully demonstrated for both a protein and an oligonucleotide, by way of which complete models were built automatically. The possibility that, through the exploitation of radiation damage, the phase problem could become less of a barrier to macromolecular crystal structure determination is discussed.

Trypsin Specificity as Elucidated by Lie Calculations, X-Ray Structures, and Association Constant Measurements

The variation in inhibitor specificity for five different amine inhibitors bound to CST, BT, and the cold‐adapted AST has been studied by use of association constant measurements, structural analysis of high‐resolution crystal structures, and the LIE method. Experimental data show that AST binds the 1BZA and 2BEA inhibitors 0.8 and 0.5 kcal/mole more strongly than BT. However, structural interactions and orientations of the inhibitors within the S1 site have been found to be virtually identical in the three enzymes studied. For example, the four water molecules in the inhibitor‐free structures of AST and BT are channeled into similar positions in the S1 site, and the nitrogen atom(s) of the inhibitors are found in two cationic binding sites denoted Position1 and Position2. The hydrophobic binding contributions for all five inhibitors, estimated by the LIE calculations, are also in the same order (−2.1 ± 0.2 kcal/mole) for all three enzymes. Our hypothesis is therefore that the observed variation in inhibitor binding arises from different electrostatic interactions originating from residues outside the S1 site. This is well illustrated by AST, in which Asp 150 and Glu 221B, despite some distance from the S1 binding site, lower the electrostatic potential of the S1 site and thus enhance substrate binding. Because the trends in the experimentally determined binding energies were reproduced by the LIE calculations after adding the contribution from long‐range interactions, we find this method very suitable for rational studies of protein–substrate interactions.

Structure of phenylalanine hydroxylase from Colwellia psychrerythraea 34H, a monomeric cold active enzyme with local flexibility around the active site and high overall stability

The characteristic of cold-adapted enzymes, high catalytic efficiency at low temperatures, is often associated with low thermostability and high flexibility. In this context, we analyzed the catalytic properties and solved the crystal structure of phenylalanine hydroxylase from the psychrophilic bacterium Colwellia psychrerythraea 34H (CpPAH). CpPAH displays highest activity with tetrahydrobiopterin (BH4) as cofactor and at 25 °C (15 °C above the optimal growth temperature). Although the enzyme is monomeric with a single l-Phe-binding site, the substrate binds cooperatively. In comparison with PAH from mesophilic bacteria and mammalian organisms, CpPAH shows elevated [S0.5](l-Phe) (= 1.1 ± 0.1 mm) and Km(BH4)(= 0.3 ± 0.1 mm), as well as high catalytic efficiency at 10 °C. However, the half-inactivation and denaturation temperature is only slightly lowered (Tm ∼ 52 °C; where Tm is half-denaturation temperature), in contrast to other cold-adapted enzymes. The crystal structure shows regions of local flexibility close to the highly solvent accessible binding sites for BH4 (Gly87/Phe88/Gly89) and l-Phe (Tyr114–Pro118). Normal mode and COREX analysis also detect these and other areas with high flexibility. Greater mobility around the active site and disrupted hydrogen bonding abilities for the cofactor appear to represent cold-adaptive properties that do not markedly affect the thermostability of CpPAH.

Atomic resolution structures of trypsin provide insight into structural radiation damage.

Radiation damage is an inherent problem in protein X-ray crystallography and the process has recently been shown to be highly specific, exhibiting features such as cleavage of disulfide bonds, decarboxylation of acidic residues, increase in atomic B factors and increase in unit-cell volume. Reported here are two trypsin structures at atomic resolution (1.00 and 0.95 A), the data for which were collected at a third-generation synchrotron (ESRF) at two different beamlines. Both trypsin structures exhibit broken disulfide bonds; in particular, the bond from Cys191 to Cys220 is very sensitive to synchrotron radiation. The data set collected at the most intense beamline (ID14-EH4) shows increased structural radiation damage in terms of lower occupancies for cysteine residues, more breakage in the six disulfide bonds and more alternate conformations. It appears that high intensity and not only the total X-ray dose is most harmful to protein crystals.

Structural basis of 5-nitroimidazole antibiotic resistance: the crystal structure of NimA from Deinococcus radiodurans.

5-Nitroimidazole-based antibiotics are compounds extensively used for treating infections in humans and animals caused by several important pathogens. They are administered as prodrugs, and their activation depends upon an anaerobic 1-electron reduction of the nitro group by a reduction pathway in the cells. Bacterial resistance toward these drugs is thought to be caused by decreased drug uptake and/or an altered reduction efficiency. One class of resistant strains, identified in Bacteroides, has been shown to carry Nim genes (NimA, -B, -C, -D, and -E), which encode for reductases that convert the nitro group on the antibiotic into a non-bactericidal amine. In this paper, we have described the crystal structure of NimA from Deinococcus radiodurans (drNimA) at 1.6 Å resolution. We have shown that drNimA is a homodimer in which each monomer adopts a β-barrel fold. We have identified the catalytically important His-71 along with the cofactor pyruvate and antibiotic binding sites, all of which are found at the monomer-monomer interface. We have reported three additional crystal structures of drNimA, one in which the antibiotic metronidazole is bound to the protein, one with pyruvate covalently bound to His-71, and one with lactate covalently bound to His-71. Based on these structures, a reaction mechanism has been proposed in which the 2-electron reduction of the antibiotic prevents accumulation of the toxic nitro radical. This mechanism suggests that Nim proteins form a new class of reductases, conferring resistance against 5-nitroimidazole-based antibiotics.

Crystal Structure of the Mobile Metallo-β-Lactamase AIM-1 from Pseudomonas aeruginosa: Insights into Antibiotic Binding and the Role of Gln157

ABSTRACT Metallo-β-lactamase (MBL) genes confer resistance to virtually all β-lactam antibiotics and are rapidly disseminated by mobile genetic elements in Gram-negative bacteria. MBLs belong to three different subgroups, B1, B2, and B3, with the mobile MBLs largely confined to subgroup B1. The B3 MBLs are a divergent subgroup of predominantly chromosomally encoded enzymes. AIM-1 (Adelaide IMipenmase 1) from Pseudomonas aeruginosa was the first B3 MBL to be identified on a readily mobile genetic element. Here we present the crystal structure of AIM-1 and use in silico docking and quantum mechanics and molecular mechanics (QM/MM) calculations, together with site-directed mutagenesis, to investigate its interaction with β-lactams. AIM-1 adopts the characteristic αβ/βα sandwich fold of MBLs but differs from other B3 enzymes in the conformation of an active site loop (residues 156 to 162) which is involved both in disulfide bond formation and, we suggest, interaction with substrates. The structure, together with docking and QM/MM calculations, indicates that the AIM-1 substrate binding site is narrower and more restricted than those of other B3 MBLs, possibly explaining its higher catalytic efficiency. The location of Gln157 adjacent to the AIM-1 zinc center suggests a role in drug binding that is supported by our in silico studies. However, replacement of this residue by either Asn or Ala resulted in only modest reductions in AIM-1 activity against the majority of β-lactam substrates, indicating that this function is nonessential. Our study reveals AIM-1 to be a subclass B3 MBL with novel structural and mechanistic features.

Is Radiation Damage Dependent on the Dose-Rate Used During Macromolecular Crystallography Data Collection?

This paper focuses on the radiation-damage effects when applying the same total X-ray dose to protein crystals at different dose rates. These experiments have been performed on both a selenomethionated protein and on bovine trypsin using dose rates that span nearly two orders of magnitude. The results show no clear dose-rate effect on the global indicators of radiation damage, but a small measurable dose-rate effect could be found when studying specific radiation damage. It is hypothesized that this observed dose-rate effect relates to differences in the steady-state free-radical concentration.

Synthetic cationic antimicrobial peptides bind with their hydrophobic parts to drug site II of human serum albumin.

BackgroundMany biologically active compounds bind to plasma transport proteins, and this binding can be either advantageous or disadvantageous from a drug design perspective. Human serum albumin (HSA) is one of the most important transport proteins in the cardiovascular system due to its great binding capacity and high physiological concentration. HSA has a preference for accommodating neutral lipophilic and acidic drug-like ligands, but is also surprisingly able to bind positively charged peptides. Understanding of how short cationic antimicrobial peptides interact with human serum albumin is of importance for developing such compounds into the clinics.ResultsThe binding of a selection of short synthetic cationic antimicrobial peptides (CAPs) to human albumin with binding affinities in the μM range is described. Competitive isothermal titration calorimetry (ITC) and NMR WaterLOGSY experiments mapped the binding site of the CAPs to the well-known drug site II within subdomain IIIA of HSA. Thermodynamic and structural analysis revealed that the binding is exclusively driven by interactions with the hydrophobic moieties of the peptides, and is independent of the cationic residues that are vital for antimicrobial activity. Both of the hydrophobic moieties comprising the peptides were detected to interact with drug site II by NMR saturation transfer difference (STD) group epitope mapping (GEM) and INPHARMA experiments. Molecular models of the complexes between the peptides and albumin were constructed using docking experiments, and support the binding hypothesis and confirm the overall binding affinities of the CAPs.ConclusionsThe biophysical and structural characterizations of albumin-peptide complexes reported here provide detailed insight into how albumin can bind short cationic peptides. The hydrophobic elements of the peptides studied here are responsible for the main interaction with HSA. We suggest that albumin binding should be taken into careful consideration in antimicrobial peptide studies, as the systemic distribution can be significantly affected by HSA interactions.

Electrostatics of mesophilic and psychrophilic trypsin isoenzymes: qualitative evaluation of electrostatic differences at the substrate binding site.

A qualitative evaluation of electrostatic features of the substrate binding region of seven isoenzymes of trypsin has been performed by using the continuum electrostatic model for the solution of the Poisson‐Boltzmann equation. The sources of the electrostatic differences among the trypsins have been sought by comparative calculations on selective charges: all charges, conserved charges, partial charges, unique cold trypsin charges, and a number of charge mutations. As expected, most of the negative potential at the S1 region of all trypsins is generated from Asp189, but the potential varies significantly among the seven trypsin isoenzymes. The three cold active enzymes included in this study possess a notably lower potential at and around the S1‐pocket compared with the warm active counterparts; this finding may be the main contribution to the increased binding affinity. The source of the differences are nonconserved charged residues outside the specificity pocket, producing electric fields at the S1‐pocket that are different in both sign and magnitude. The surface charges of the mesophilic trypsins generally induce the S1 pocket positively, whereas surface charges of the cold trypsins produce a negative electric field of this region. Calculations on mutants, where charged amino acids were substituted between the trypsins, showed that mutations in Loop2 (residues 221B and 224) and residue 175, in particular, were responsible for the low potential of the cold enzymes. Proteins 2000;40:207–217.