Short hydrogen bonds (H-bonds) have been proposed to play key functional roles in several proteins. The location of the proton in short H-bonds is of central importance, as proton delocalization is a ...defining feature of low-barrier hydrogen bonds (LBHBs). Experimentally determining proton location in H-bonds is challenging. Here, bond length analysis of atomic (1.15–0.98 Å) resolution X-ray crystal structures of the human protein DJ-1 and its bacterial homologue, YajL, was used to determine the protonation states of H-bonded carboxylic acids. DJ-1 contains a buried, dimer-spanning 2.49 Å H-bond between Glu15 and Asp24 that satisfies standard donor–acceptor distance criteria for a LBHB. Bond length analysis indicates that the proton is localized on Asp24, excluding a LBHB at this location. However, similar analysis of the Escherichia coli homologue YajL shows both residues may be protonated at the H-bonded oxygen atoms, potentially consistent with a LBHB. A Protein Data Bank-wide screen identifies candidate carboxylic acid H-bonds in approximately 14% of proteins, which are typically short ⟨d O–O⟩ = 2.542(2) Å. Chemically similar H-bonds between hydroxylated residues (Ser/Thr/Tyr) and carboxylates show a trend of lengthening O–O distance with increasing H-bond donor pK a. This trend suggests that conventional electronic effects provide an adequate explanation for short, charge-assisted carboxylic acid–carboxylate H-bonds in proteins, without the need to invoke LBHBs in general. This study demonstrates that bond length analysis of atomic resolution X-ray crystal structures provides a useful experimental test of certain candidate LBHBs.
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Determination of a protein-ligand complex structure is essential in many areas of structural biology. Details of the interactions between protein and a small molecule ligand often ...represent major findings from a crystal structure. Thorough validation of interpretation of such structural data is particularly important given high expectation of confirming prior experimental findings regarding targeted protein-ligand interaction. Modern methods of ligand validation are discussed and illustrated.
As a result of substantial instrumental automation and the continuing improvement of software, crystallographic studies of biomolecules are conducted by non‐experts in increasing numbers. While ...improved validation almost ensures that major mistakes in the protein part of structure models are exceedingly rare, in ligand–protein complex structures, which in general are most interesting to the scientist, ambiguous ligand electron density is often difficult to interpret and the modelled ligands are generally more difficult to properly validate. Here, (i) the primary technical reasons and potential human factors leading to problems in ligand structure models are presented; (ii) the most common categories of building errors or overinterpretation are classified; (iii) a few instructive and specific examples are discussed in detail, including an electron‐density‐based analysis of ligand structures that do not contain any ligands; (iv) means of avoiding such mistakes are suggested and the implications for database validity are discussed and (v) a user‐friendly software tool that allows non‐expert users to conveniently inspect ligand density is provided.
It is demonstrated that the crystallographic models of macromolecules may appear to diverge upon extended refinement against experimental data. Two regimes are identified for this phenomenon. ...Firstly, at higher resolution the apparent instability of the resulting models is shown to originate from the relatively small fraction of disordered atoms present in the initial model. Secondly, at lower resolution additional refinement instability may arise from insufficiently strong geometry restraints. The convergence of crystallographic refinement is proposed as one of the possible criteria in selecting a specific refinement strategy and in model validation.
A flow fluorometric approach to study cationic lipoid–DNA complexes is presented. The approach uses standard flow cytometry equipment and common fluorescent dyes (BODIPY and ethidium homodimer-2) to ...detect both lipoid and DNA content in individual particles. In addition, a procedure that allows determination of whether or not liposomes remain intact is described. The procedure is based on monitoring the retention of a polar tracer that has been preloaded into its aqueous compartment. Sample preparation, instrument setup, data analysis, and methodological limitations are described. Applications of the procedure to cationic lipoid–DNA complexes are described, and illustrations are given for the determination of how the lipoid content, composition, and structure of individual lipoplexes in a population evolve over time, starting at about 1
min after DNA and vesicles are mixed. Analogous procedures can be applied to other heterogeneous particles and supramolecular structures.
Antibodies against cocaine and other drugs of abuse are the basis for diagnostic tests for the presence of those drugs in human serum. The 1.7
Å resolution crystal structure of the anti-cocaine ...monoclonal antibody M82G2 in complex with cocaine is presented. This structure determination was undertaken to establish the stereochemical features in the antibody binding site that confer specificity for cocaine, and as part of an ongoing project to understand the rules that govern molecular recognition. The cocaine-binding site can be characterized topologically as a narrow groove on the protein surface. The antibody utilizes water-mediated hydrogen bonding, and cation–π and stacking (π–π) interactions to provide specificity. Comparison with the previously published structure of the anti-cocaine antibody GNC92H2 shows that binding of a small ligand can be achieved in diverse ways, both in terms of a binding site structure/topology and protein–ligand interactions.
Therapeutic solutions to combat the Clostridioides difficile infection (CDI) are high in‐demand due to antibiotic resistance and limited treatment options. C. difficile transferase (CDT) binary toxin ...is associated with hypervirulence, in addition to the major clostridial toxins. CDT consists of an enzymatic component (CDTa), and a pore‐forming binding component (CDTb). Cytotoxicity of the host cell is initiated by the formation of CDTa‐CDTb binary toxin complex and subsequent conformational shift in the CDTb to enforce translocation of the CDTa into the cell. This results in destruction of the host cell cytoskeleton through ADP‐ribosylation of the actin filaments.
Molecular level characterization of full length active wild‐type CDTb by Cryogenic Electron Microscopy (CryoEM) revealed existence of two novel di‐heptamer units in the absence of full‐length CDTa. The ‘pore’ state is represented by extended β‐barrel assembly in the AsymCDTb structure and the ‘pre‐pore’ state or the SymCDTb form consisted identical heptamer domains. Detailed analysis of the structure depicted the presence of different domains including two receptor binding domains (RBD). The RBD1 domain represents a discrete non‐homologous structure that was not identified in other binary toxins and the RBD2 domain is crucial for di‐heptamer assembly and physiological activity of the toxin.
A unique calcium‐binding site in RBD1 in the X‐ray crystal structure of AsymCDTb pinpointed the significant role of calcium in the structural stability and the protein folding mechanisms. Historically, potent calcium‐containing therapeutic agents have been able to neutralize the activity of large clostridial toxins. Therefore, we explored the structural features of the CDTbD623/734A double mutant which disrupted the calcium binding site in the RBD1. The 2D‐structural details revealed unique conformational changes in the heptameric organization of the mutant which indicated potential disturbances to the formation of β‐barrel. We compared the toxicity of wild type CDTb and CDTbD623/734A in the presence of CDTa using a Vero cell assay. CDTbD623/734A showed a significant decrease in toxicity (TC50 CDTb 80±6pM; CDTbD623/734A 637±20pM), supporting the idea that this novel calcium‐binding site is required for function.
Calcium plays a pivotal role in the structure‐activity relationship and toxin machinery. Therefore, ablation of the divalent interaction and targeting the binding site stand out as promising strategies in the drug development for CDI. Unraveling the distinctive conformational features in different mutant constructs of the binary toxin can be beneficial in fine‐tuning these potential therapeutic agents.
Free energies of both urea and thermal denaturation have been measured for three pairs of one- and two-repeat fragments, cloned in tandem from the cytoskeletal protein, α-spectrin, from chicken brain ...to ascertain whether one- and two-repeat fragments are equally stable. One- and two-repeat fragments of each pair were designed with the same N-terminus, whereas the C-terminus of the two-repeat fragment was 106 residues or the length of one repeat downstream from that of the one-repeat fragment. The averaged free energies of urea and thermal denaturation of the paired fragments, (R16)00 and (R16R17)00, (R16)0+3 and (R16R17)0+3, and (R16)+8 - 4 and (R16R17)+8 - 4 subscripts represent the N- and C-terminal positions with “00” referring to the N- and C-termini defining a repeat according to X-ray crystal structures of two repeat fragments Grum, V. L., Li, D., MacDonald, R. I., and Mondragón, A. (1999) Cell 98, 523−535 and “+” and “−” referring to positions upstream and downstream therefrom, respectively, increased from 3.7 ± 0.4 kcal/mol for (R16)00, 3.7 ± 0.5 kcal/mol for (R16)0+3, 4.4 ± 0.4 kcal/mol for (R16)+8 - 4, 6.2 ± 0.6 kcal/mol for (R16R17)+8 - 4, 8.3 ± 0.4 kcal/mol for (R16R17)00 to 9.9 ± 1.0 kcal/mol for (R16R17)0+3. Thus, the two-repeat fragment of each pair was significantly more thermodynamically stable than the single repeat by both urea and thermal denaturation. Differences in phasing among single repeats did not have the same effect as the same differences in phasing among two-repeat fragments. Addition of nine residues to the C-terminus of (R16R17)00 yielded a free energy of unfolding of 7.9 ± 0.8 kcal/mol, whereas addition of seven residues to the C-terminus of (R16)+8 - 4 yielded a free energy of unfolding of 5.9 ± 0.3 kcal/mol.
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