Marilyn D. Yoder

Unusual structural features in the parallel β-helix in pectate lyases

Abstract Background: A new type of domain structure, an all parallel β class, has recently been observed in two pectate lyases, PelC and PelE. The atomic models have been analyzed to determine whether the new tertiary fold exhibits unusual structural features. Results: The polypeptide backbone exhibits no new types of secondary structural elements. However, novel features occur in the amino acid side chain interactions. The side chain atoms form linear stacks that include asparagine ladders, serine stacks, aliphatic stacks, and ringed-residue stacks. A new type of β-sandwich between parallel β-sheets is observed with properties that are more characteristic of antiparallel β-sheets. Conclusion: An analysis of the PelC and PelE structures, belonging to an all parallel β structural class, reveals novel amino acid side chain interactions, a new type of β-sandwich and an atypical amino acid composition of parallel β-sheets. The findings are relevant to three-dimensional structural predictions.

Functional Implications of Structure-Based Sequence Alignment of Proteins in the Extracellular Pectate Lyase Superfamily

Pectate lyases are plant virulence factors that degrade the pectate component of the plant cell wall. The enzymes share considerable sequence homology with plant pollen and style proteins, suggesting a shared structural topology and possibly functional relationships as well. The three-dimensional structures of two Erwinia chrysanthemi pectate lyases, C and E, have been superimposed and the structurally conserved amino acids have been identified. There are 232 amino acids that superimpose with a root-mean-square deviation of 3 A or less. These amino acids have been used to correct the primary sequence alignment derived from evolution-based techniques. Subsequently, multiple alignment techniques have allowed the realignment of other extracellular pectate lyases as well as all sequence homologs, including pectin lyases and the plant pollen and style proteins. The new multiple sequence alignment reveals amino acids likely to participate in the parallel [beta] helix motif, those involved in binding Ca2+, and those invariant amino acids with potential catalytic properties. The latter amino acids cluster in two well-separated regions on the pectate lyase structures, suggesting two distinct enzymatic functions for extracellular pectate lyases and their sequence homologs.

The Three-Dimensional Structure of Pectate Lyase E, a Plant Virulence Factor from Erwinia chrysanthemi.

The three-dimensional structure of pectate lyase E (PelE) has been determined by crystallographic techniques at a resolution of 2.2 A. The model includes all 355 amino acids but no solvent, and refines to a crystallographic refinement factor of 20.6%. The polypeptide backbone folds into a large right-handed cylinder, termed a parallel [beta] helix. Loops of various sizes and conformations protrude from the central helix and probably confer function. A putative Ca2+-binding site as well as two cationic sites have been deduced from the location of heavy atom derivatives. Comparison of the PelE and recently determined pectate lyase C (PelC) structures has led to identification of a putative polygalacturonate-binding region in PelE. Structural differences relevant to differences in the enzymatic mechanism and maceration properties of PelE and PelC have been identified. The comparative analysis also reveals a large degree of structural conservation of surface loops in one region as well as an apparent aromatic specificity pocket in the amino-terminal branch. Also discussed is the sequence and possible functional relationship of the pectate lyases with pollen and style plant proteins.

The Refined Three-Dimensional Structure of Pectate Lyase C from Erwinia chrysanthemi at 2.2 Angstrom Resolution (Implications for an Enzymatic Mechanism).

The crystal structure of pectate lyase C (EC 4.2.2.2) from the enterobacterium Erwinia chrysanthemi (PelC) has been refined by molecular dynamics techniques to a resolution of 2.2 A to an R factor of 17.97%. The final model consists of 352 of the total 353 amino acids and 114 solvent molecules. The root-mean-square deviation from ideality is 0.009 A for bond lengths and 1.768[deg] for bond angles. The structure of PelC bound to the lanthanide ion lutetium, used as a calcium analog, has also been refined. Lutetium inhibits the enzymatic activity of the protein, and in the PelC-lutetium structure, the ion binds in the putative calcium-binding site. Five side-chain atoms form ligands to the lutetium ion. An analysis of the atomic-level model of the two protein structures reveals possible implications for the enzymatic mechanism of the enzyme.

The Refined Three-Dimensional Structure of Pectate Lyase E from Erwinia chrysanthemi at 2.2 A Resolution.

The crystal structure of pectate lyase E (PelE; EC 4.2.2.2) from the enterobacteria Erwinia chrysanthemi has been refined by molecular dynamics techniques to a resolution of 2.2 A and an R factor (an agreement factor between observed structure factor amplitudes) of 16.1%. The final model consists of all 355 amino acids and 157 water molecules. The root-mean-square deviation from ideality is 0.009 A for bond lengths and 1.721[deg] for bond angles. The structure of PelE bound to a lanthanum ion, which inhibits the enzymatic activity, has also been refined and compared to the metal-free protein. In addition, the structures of pectate lyase C (PelC) in the presence and absence of a lutetium ion have been refined further using an improved algorithm for identifying waters and other solvent molecules. The two putative active site regions of PelE have been compared to those in the refined structure of PelC. The analysis of the atomic details of PelE and PelC in the presence and absence of lanthanide ions provides insight into the enzymatic mechanism of pectate lyases.

Parallel β-domains: a new fold in protein structures

Abstract A new type of structural domain, composed of parallel β-strands folded into a coiled structure, has been observed in several protein structures within the past year. An analysis of the basic motif indicates that there are two distinct types, with variations likely to be discovered in the future.

Bulk preparation and crystallization of the Escherichia coli elongation factor Tu-Ts complex☆

A simple procedure for the preparation of 10-500 mg of the Escherichia coli elongation Tu-Ts complex is described. The protocol is based on the separate purification and quantitation of EF-Tu-GDP and EF-Ts, followed by mixing of equimolar amounts of each protein and removal of the displaced GDP by dialysis. Single crystals grown from the final product have been analyzed by X-ray diffraction techniques. The procedure is also applicable to the bulk preparation and crystallization of the trypsin-modified Tu-Ts complex. Quantitation of the elongation factors by three methods is presented.

Affinity purification of aminoacyl-tRNA☆

A procedure for separating Escherichia coli aminoacyl-tRNA from unacylated tRNA or components of the aminoacylation reaction, thereby achieving an aminoacyl-tRNA product with a very high specific activity, is described. The method utilizes the specific recognition of aminoacyl-tRNA for E. coli protein synthesis elongation factor Tu which has been immobilized on an affinity matrix. The application of the affinity procedure as a means of purifying a single aminoacyl-tRNA from an unfractionated mixture of tRNAs is also discussed.

An improved bulk purification method for Escherichia coli elongation factor, Ts☆

A bulk purification procedure has been designed to maximize the yield of Escherichia coli elongation factor, Ts, with a minimum of effort and time. The enzyme purification is achieved by DEAE-Sepharose and elongation factor Tu-affinity chromatographies. The typical yield is 150 mg/kg of E. coli (B) cells.