Johan Kördel

The serum albumin-binding domain of streptococcal protein G is a three-helical bundle: a heteronuclear NMR study

Streptococcal protein G (SPG) is a cell surface receptor protein with a multiple domain structure containing tandem repeats of serum albumin‐binding domains (ABD) and immunoglobulin‐binding domains (IgBD). In this paper, we have analysed the fold of ABD. Far‐UV circular dichroism analysis of ABD indicates high helical content (56%). Based on an analysis of nuclear magnetic resonance 13C secondary chemical shifts, sequential and short‐range NOEs, and a few key nuclear Overhauser effects, we conclude that the ABD is a three‐helix bundle. The structure of the ABD is, thus, quite different from the IgBD of protein G [Gronenborn, A.M. et al. (1991) Science 253, 657–661]. This strongly suggests that the ABD and the IgBD of SPG have evolved independently from each other. However, the fold of ABD is similar to that of the IgBD of staphylococcal protein A, possibly indicating a common evolutionary ancestor, despite the lack of sequence homology.

Nuclear magnetic resonance studies of the internal dynamics in apo, (Cd2+)1 and (Ca2+)2 Calbindin D9k: The rates of amide proton exchange with solvent

The backbone dynamics of the EF-hand Ca(2+)-binding protein, calbindin D9k, has been investigated in the apo, (Cd2+)1 and (Ca2+)2 states by measuring the rate constants for amide proton exchange with solvent. 15N-1H correlation spectroscopy was utilized to follow direct 1H-->2H exchange of the slowly exchanging amide protons and to follow indirect proton exchange via saturation transfer from water to the rapidly exchanging amide protons. Plots of experimental rate constants versus intrinsic rate constants have been analyzed to give qualitative insight into the opening modes of the protein that lead to exchange. These results have been interpreted within the context of a progressive unfolding model, wherein hydrophobic interactions and metal chelation serve to anchor portions of the protein, thereby damping fluctuations and retarding amide proton exchange. The addition of Ca2+ or Cd2+ was found to retard the exchange of many amide protons observed to be in hydrogen-bonding environments in the crystal structure of the (Ca2+)2 state, but not of those amide protons that were not involved in hydrogen bonds. The largest changes in rate constant occur for residues in the ion-binding loops, with substantial effects also found for the adjacent residues in helices I, II and III, but not helix IV. The results are consistent with a reorganization of the hydrogen-bonding networks in the metal ion-binding loops, accompanied by a change in the conformation of helix IV, as metal ions are chelated. Further analysis of the results obtained for the three states of metal occupancy provides insight into the nature of the changes in conformational fluctuations induced by ion binding.

Characterization of Ligand Binding of a Soluble Human Insulin-like Growth Factor I Receptor Variant Suggests a Ligand-induced Conformational Change

Details of the signal transduction mechanisms of the tyrosine kinase family of growth factor receptors remain elusive. In this work, we describe an extensive study of kinetic and thermodynamic aspects of growth factor binding to a soluble extracellular human insulin-like growth factor-I receptor (sIGF-IR) variant. The extracellular receptor domains were produced fused to an IgG-binding protein domain (Z) in transfected human 293 cells as a correctly processed secreted α-β′-Z dimer. The receptor was purified using IgG affinity chromatography, rendering a pure and homogenous protein in yields from 1 to 5 mg/liter of conditioned cell media. Biosensor technology (BIAcore) was applied to measure the insulin-like growth factor-I (IGF-I), des(1-3)IGF-I, insulin-like growth factor-II, and insulin ligand binding rate constants to the immobilized IGF-IR-Z. The association equilibrium constant, Ka, for the IGF-I interaction is determined to 2.8 × 108 M−1 (25 °C). Microcalorimetric titrations on IGF-I/IGF-IR-Z were performed at three different temperatures (15, 25, and 37 °C) and in two different buffer systems at 25 °C. From these measurements, equilibrium constants for the 1:1 (IGF-I:(α-β′-Z)2) receptor complex in solution are deduced to 0.96 × 108 M−1 (25 °C). The determined heat capacity change for the process is large and negative, −(1)0.51 kcal (K mol)−1. Further, the entropy change (ΔS) at 25 °C is large and negative. Far- and near-UV circular dichroism measurements display significant changes over the entire wavelength range upon binding of IGF-I to IGF-IR-Z. These data are all consistent with a significant change in structure of the system upon IGF-I binding.

Comparative strutural analysis of the calcium free and bound states of the calcium regulatory protein calbindin D9K

The solution structure of apo calbindin D9K, a member of the calmodulin superfamily of calcium-binding regulatory proteins, has been investigated by 1H nuclear magnetic resonance spectroscopy and the results compared with a corresponding study of the calcium-loaded protein. On the basis of complete sequence-specific assignments, characteristic patterns of short proton-proton distances have been identified in two-dimensional nuclear Overhauser effect spectra, allowing the elements of secondary structure to be determined. It is found that four helices and a short section of antiparallel beta-sheet are present regardless of the calcium content of the protein. In addition, a preliminary analysis of the long-range nuclear Overhauser effects shows that the global folding patterns are the same and that the tertiary structures of the apo protein is very similar to that of the calcium-loaded protein. These results are in stark contrast to a number of very substantial changes in 1H chemical shift. Preliminary studies of protein dynamics show some very large differences in flexibility and internal mobility. This suggests that protein dynamics may play a role more important than was initially realized in the function of calbindin D9K and other homologous calcium-binding regulatory proteins.

Dissection of Calbindin D9k into two Ca2+-binding subdomains by a combination of mutagenesis and chemical cleavage

Calbindin D9k is a 75‐residue globular protein made up of two Ca2+‐binding subdomains of the EF‐hand type. In order to examine the subdomains independently, a method was devised to selectively cleave the loop between them. Using site‐directed mutagenesis, a unique methionine was substituted for Pro43 in the loop, thus allowing cleavage using cyanogen bromide. Agarose gel electrophoresis shows that the fragments have a high affinity for one another, although less so in the absence of calcium.1H‐NMR spectra of the fragments indicate that the structures of the heterodimers are changed little from that of the intact protein. However, the Ca2+ binding constants of the individual subdomains are several orders of magnitude lower than for the corresponding sites in the uncleaved protein.

Protein solution structure calculations in solution: Solvated molecular dynamics refinement of calbindin D9k

The three-dimensional solution structures of proteins determinedwith NMR-derived constraints are almost always calculated in vacuo. Thesolution structure of (Ca2+)_2-calbindinD9k has been redetermined by new restrained molecular dynamics(MD) calculations that include Ca2+ ions and explicit solventmolecules. Four parallel sets of MD refinements were run to provide accuratecomparisons of structures produced in vacuo, in vacuo withCa2+ ions, and with two different protocols in a solvent bathwith Ca2+ ions. The structural ensembles were analyzed interms of structural definition, molecular energies, packing density,solvent-accessible surface, hydrogen bonds, and the coordination of calciumions in the two binding loops. Refinement including Ca2+ ionsand explicit solvent results in significant improvements in the precisionand accuracy of the structure, particularly in the binding loops. Theseresults are consistent with results previously obtained in free MDsimulations of proteins in solution and show that the rMD refinedNMR-derived solution structures of proteins, especially metalloproteins, canbe significantly improved by these strategies.

15N NMR assignments and chemical shift analysis of uniformly labeled 15N calbindin D9k in the apo, (Cd2+)1 and (Ca2+)2 states

15N has been uniformly incorporated into the EF‐hand Ca2+‐binding protein calbindin D9k so that heteronuclear experiments can be used to further characterize the structure and dynamics of the apo, (Cd2+)1 and (Ca2+)2 states of the protein. The15N NMR resonances were assigned by 2D15N‐resolved1H experiments, which also allowed the identification of a number of sequential and medium‐range1H 1H contacts that are obscured by chemical shift degeneracy in homonuclear experiments. The15N chemical shifts are analyzed with respect to correlations with protein secondary structure. In addition, the changes in15N chemical shift found for the apo→(Cd22+)1→(Ca2+)2 binding sequence confirm that the effects on the protein are mainly associated with chelation of the first ion.

Peptidyl-prolyl cis-trans isomerase does not affect the Pro-43 cis-trans isomerization rate in folded calbindin D9k.

The calcium‐binding protein calbindin D9k has previously been shown to exist in two folded forms only differing in the proline cis‐trans isomerism of the Gly‐42‐Pro‐43 amide bond. This bond is located in a flexible loop connecting the two EF‐hand Ca2+ sites. Calbindin D9k therefore constitutes a unique test case for investigating if the recently discovered enzyme peptidyl‐prolyl cis‐trans isomerase (PPIase) can affect the cis‐trans exchange rate in a folded protein. The 1H NMR saturation transfer technique has been used to measure the rate of interconversion between the cis and trans forms of calbindin in the presence of PPIase (PPIase:calbindin concentration ratio 1:10) at 35°C. No rate enhancement could be detected.

Analysis of myo-inositol phosphates by 2D 1H-n.m.r. spectroscopy

Abstract 2D 1 H-N.m.r. spectroscopy applied to phosphorylated myo -inositols can indicate the number and the sites of phosphorylation, and up to three isomers can be completely analyzed simultaneously. The resonance of the sole equatorial proton (H-2) is shifted furthest downfield (to ∼4.7 p.p.m.) when C-2 is phosphorylated; when C-5 is unphosphorylated, the H-5 resonance has the lowest chemical shift (∼ 3.3 p.p.m.). The number of sites of phosphorylation can be determined from the sum of the chemical shifts for the resonances in a given myo -inositol phosphate and, based on the data presented, an algorithm can be constructed that will identify any myo -inositol phosphate on the basis of the chemical shifts.

PROTEIN ENGINEERING AND STRUCTURE/FUNCTION RELATIONS IN BOVINE CALBINDIN D9k

In an attempt to elucidate the relationships between structure, dynamics and function in the calmodulin superfamily of Ca2+-binding intracellular proteins we have undertaken a detailed study of bovine calbindin D9k. This protein has a size (Mr≃8,500) and tertiary structure similar to that of the globular domains of calmodulin and troponin C and binds two Ca2+ -ions strongly (K ≃107 - 108 M-1, depending on the ionic strength). The schematic structure of the molecule is shown in figure 1.