Karl Volz

Structure of dual function iron regulatory protein 1 complexed with ferritin IRE-RNA.

Iron regulatory protein 1 (IRP1) binds iron-responsive elements (IREs) in messenger RNAs (mRNAs), to repress translation or degradation, or binds an iron-sulfur cluster, to become a cytosolic aconitase enzyme. The 2.8 angstrom resolution crystal structure of the IRP1:ferritin H IRE complex shows an open protein conformation compared with that of cytosolic aconitase. The extended, L-shaped IRP1 molecule embraces the IRE stem-loop through interactions at two sites separated by ∼30 angstroms, each involving about a dozen protein:RNA bonds. Extensive conformational changes related to binding the IRE or an iron-sulfur cluster explain the alternate functions of IRP1 as an mRNA regulator or enzyme.

Crystal structure of human PEDF, a potent anti-angiogenic and neurite growth-promoting factor.

Pigment epithelium-derived factor (PEDF), a noninhibitory member of the serpin superfamily, is the most potent inhibitor of angiogenesis in the mammalian ocular compartment. It also has neurotrophic activity, both in the retina and in the central nervous system, and is highly up-regulated in young versus senescent fibroblasts. To provide a structural basis for understanding its many biological roles, we have solved the crystal structure of glycosylated human PEDF to 2.85 Å. The structure revealed the organization of possible receptor and heparin-binding sites, and showed that, unlike any other previously characterized serpin, PEDF has a striking asymmetric charge distribution that might be of functional importance. These results provide a starting point for future detailed structure/function analyses into possible mechanisms of PEDF action that could lead to development of therapeutics against uncontrolled angiogenesis.

Crystal Structures of CheY Mutants Y106W and T87I/Y106W CheY ACTIVATION CORRELATES WITH MOVEMENT OF RESIDUE 106

Position 106 in CheY is highly conserved as an aromatic residue in the response regulator superfamily. In the structure of the wild-type, apo-CheY, Tyr106 is a rotamer whose electron density is observed in both the inside and the outside positions. In the structure of the T87I mutant of CheY, the threonine to isoleucine change at position 87 causes the side chain of Tyr106 to be exclusively restricted to the outside position. In this report we demonstrate that the T87I mutation causes cells to be smooth swimming and non-chemotactic. We also show that another CheY mutant, Y106W, causes cells to be more tumbly than wild-type CheY, and impairs chemotaxis. In the structure of Y106W, the side chain of Trp106 stays exclusively in the inside position. Furthermore, a T87I/Y106W double mutant, which confers the same phenotype as T87I, restricts the side chain of Trp106 to the outside position. The results from these behavioral and structural studies indicate that the rotameric nature of the Tyr106 residue is involved in activation of the CheY molecule. Specifically, CheYs signaling ability correlates with the conformational heterogeneity of the Tyr106 side chain. Our data also suggest that these mutations affect the signal at an event subsequent to phosphorylation.

Canonical inhibitor-like interactions explain reactivity of alpha1-proteinase inhibitor Pittsburgh and antithrombin with proteinases

The serpin antithrombin is a slow thrombin inhibitor that requires heparin to enhance its reaction rate. In contrast, α1-proteinase inhibitor (α1PI) Pittsburgh (P1 Met → Arg natural variant) inhibits thrombin 17 times faster than pentasaccharide heparin-activated antithrombin. We present here x-ray structures of free and S195A trypsin-bound α1PI Pittsburgh, which show that the reactive center loop (RCL) possesses a canonical conformation in the free serpin that does not change upon binding to S195A trypsin and that contacts the proteinase only between P2 and P2′. By inference from the structure of heparin cofactor II bound to S195A thrombin, this RCL conformation is also appropriate for binding to thrombin. Reaction rates of trypsin and thrombin with α1PI Pittsburgh and antithrombin and their P2 variants show that the low antithrombin-thrombin reaction rate results from the antithrombin RCL sequence at P2 and implies that, in solution, the antithrombin RCL must be in a similar canonical conformation to that found here for α1PI Pittsburgh, even in the nonheparin-activated state. This suggests a general, limited, canonical-like interaction between serpins and proteinases in their Michaelis complexes.

The functional duality of iron regulatory protein 1

Iron homeostasis in animal cells is controlled post-transcriptionally by the iron regulatory proteins IRP1 and IRP2. IRP1 can assume two different functions in the cell, depending on conditions. During iron scarcity or oxidative stress, IRP1 binds to mRNA stem-loop structures called iron responsive elements (IREs) to modulate the translation of iron metabolism genes. In iron-rich conditions, IRP1 binds an iron-sulfur cluster to function as a cytosolic aconitase. This functional duality of IRP1 connects the translational control of iron metabolizing proteins to cellular iron levels. The recently determined structures of IRP1 in both functional states reveal the large-scale conformational changes required for these mutually exclusive roles, providing new insights into the mechanisms of IRP1 interconversion and ligand binding.

Canonical inhibitor-like interactions explain reactivity of α-PI Pittsburgh and antithrombin with proteinases

The serpin antithrombin is a slow thrombin inhibitor that requires heparin to enhance its reaction rate. In contrast, α1-proteinase inhibitor (α1PI) Pittsburgh (P1 Met → Arg natural variant) inhibits thrombin 17 times faster than pentasaccharide heparin-activated antithrombin. We present here x-ray structures of free and S195A trypsin-bound α1PI Pittsburgh, which show that the reactive center loop (RCL) possesses a canonical conformation in the free serpin that does not change upon binding to S195A trypsin and that contacts the proteinase only between P2 and P2′. By inference from the structure of heparin cofactor II bound to S195A thrombin, this RCL conformation is also appropriate for binding to thrombin. Reaction rates of trypsin and thrombin with α1PI Pittsburgh and antithrombin and their P2 variants show that the low antithrombin-thrombin reaction rate results from the antithrombin RCL sequence at P2 and implies that, in solution, the antithrombin RCL must be in a similar canonical conformation to that found here for α1PI Pittsburgh, even in the nonheparin-activated state. This suggests a general, limited, canonical-like interaction between serpins and proteinases in their Michaelis complexes.

Crystal Structure of Human Maspin, a Serpin with Antitumor Properties REACTIVE CENTER LOOP OF MASPIN IS EXPOSED BUT CONSTRAINED

Maspin, a member of the serpin superfamily, has tumor suppressing activity against breast and prostate cancer. Maspin inhibits tumor growth by blocking cell invasion, and its reactive center loop (RCL) is thought to mediate this activity. To understand this function on the molecular level, we have solved the three-dimensional structure of Maspin to 3.1 Å resolution. The molecular structure shows the characteristic features of the serpin fold, but the RCL of Maspin is unique in length, composition, and placement. Although the RCL of Maspin is accessible and cleavable by some proteinases, it functions in the uncleaved, constrained conformation observed here. These structural results will contribute to our understanding of the mechanism by which Maspin suppresses tumors.

Pigment Epithelium-Derived Factor (PEDF), a Serpin with Potent Anti-Angiogenic and Neurite Outgrowth-promoting Properties

Abstract Pigment epitheliumderived factor is a member of the serpin superfamily of proteins, but one that lacks inhibitory properties against either serine or cysteine proteinases. Nevertheless it possesses a number of physiological properties that make it a potentially important protein in regulation of angiogenesis, in neuronal cell survival and in protection of neurons from neurotoxic agents. It is also a protein that is highly up regulated in the G0 phase of earlypassage cells compared with rapidly proliferating cells or senescent cells, and so is also linked to both the cell cycle and cell senescence. The determination of a high resolution Xray crystal structure of native PEDF provides insight into regions of the protein that may be involved in one or more of these functions.

Crystallization and preliminary X-ray diffraction analysis of iron regulatory protein 1 in complex with ferritin IRE RNA

Iron regulatory protein 1 (IRP1) is a bifunctional protein with activity as an RNA-binding protein or as a cytoplasmic aconitase. Interconversion of IRP1 between these mutually exclusive states is central to cellular iron regulation and is accomplished through iron-responsive assembly and disassembly of a [4Fe-4S] cluster. When in its apo form, IRP1 binds to iron responsive elements (IREs) found in mRNAs encoding proteins of iron storage and transport and either prevents translation or degradation of the bound mRNA. Excess cellular iron stimulates the assembly of a [4Fe-4S] cluster in IRP1, inhibiting its IRE-binding ability and converting it to an aconitase. The three-dimensional structure of IRP1 in its different active forms will provide details of the interconversion process and clarify the selective recognition of mRNA, Fe-S sites and catalytic activity. To this end, the apo form of IRP1 bound to a ferritin IRE was crystallized. Crystals belong to the monoclinic space group P2(1), with unit-cell parameters a = 109.6, b = 80.9, c = 142.9 A, beta = 92.0 degrees. Native data sets have been collected from several crystals with resolution extending to 2.8 A and the structure has been solved by molecular replacement.

Probing the Role of Protein Surface Charge in the Activation of PrfA, the Central Regulator of Listeria monocytogenes Pathogenesis

Listeria monocytogenes is a food-borne intracellular bacterial pathogen capable of causing serious human disease. L. monocytogenes survival within mammalian cells depends upon the synthesis of a number of secreted virulence factors whose expression is regulated by the transcriptional activator PrfA. PrfA becomes activated following bacterial entry into host cells where it induces the expression of gene products required for bacterial spread to adjacent cells. Activation of PrfA appears to occur via the binding of a small molecule cofactor whose identity remains unknown. Electrostatic modeling of the predicted PrfA cofactor binding pocket revealed a highly positively charged region with two lysine residues, K64 and K122, located at the edge of the pocket and another (K130) located deep within the interior. Mutational analysis of these residues indicated that K64 and K122 contribute to intracellular activation of PrfA, whereas a K130 substitution abolished protein activity. The requirement of K64 and K122 for intracellular PrfA activation could be bypassed via the introduction of the prfA G145S mutation that constitutively activates PrfA in the absence of cofactor binding. Our data indicate that the positive charge of the PrfA binding pocket contributes to intracellular activation of PrfA, presumably by facilitating binding of an anionic cofactor.