Histidine Hydrogen-Deuterium Exchange Mass Spectrometry for Probing the Microenvironment of Histidine Residues in Dihydrofolate Reductase

Histidine Hydrogen-Deuterium Exchange Mass Spectrometry (His-HDX-MS) determines the HDX rates at the imidazole C(2)-hydrogen of histidine residues. This method provides not only the HDX rates but also the pK(a) values of histidine imidazole rings. His-HDX-MS was used to probe the microenvironment of histidine residues of E. coli dihydrofolate reductase (DHFR), an enzyme proposed to undergo multiple conformational changes during catalysis.Using His-HDX-MS, the pK(a) values and the half-lives (t(1/2)) of HDX reactions of five histidine residues of apo-DHFR, DHFR in complex with methotrexate (DHFR-MTX), DHFR in complex with MTX and NADPH (DHFR-MTX-NADPH), and DHFR in complex with folate and NADP+ (DHFR-folate-NADP+) were determined. The results showed that the two parameters (pK(a) and t(1/2)) are sensitive to the changes of the microenvironment around the histidine residues. Although four of the five histidine residues are located far from the active site, ligand binding affected their pK(a), t(1/2) or both. This is consistent with previous observations of ligand binding-induced distal conformational changes on DHFR. Most of the observed pK(a) and t(1/2) changes could be rationalized using the X-ray structures of apo-DHFR, DHFR-MTX-NADPH, and DHFR-folate-NADP+. The availability of the neutron diffraction structure of DHFR-MTX enabled us to compare the protonation states of histidine imidazole rings.Our results demonstrate the usefulness of His-HDX-MS in probing the microenvironments of histidine residues within proteins

The <i>Aspergillus fumigatus pkcA</i>^G579R Mutant Is Defective in the Activation of the Cell Wall Integrity Pathway but Is Dispensable for Virulence in a Neutropenic Mouse Infection Model

<div><p><i>Aspergillus fumigatus</i> is an opportunistic human pathogen, which causes the life-threatening disease, invasive pulmonary aspergillosis. In fungi, cell wall homeostasis is controlled by the conserved <u>C</u>ell <u>W</u>all <u>I</u>ntegrity (CWI) pathway. In <i>A</i>. <i>fumigatus</i> this signaling cascade is partially characterized, but the mechanisms by which it is activated are not fully elucidated. In this study we investigated the role of protein kinase C (PkcA) in this signaling cascade. Our results suggest that <i>pkcA</i> is an essential gene and is activated in response to cell wall stress. Subsequently, we constructed and analyzed a non-essential <i>A</i>. <i>fumigatus pkcA</i>^G579R mutant, carrying a Gly579Arg substitution in the PkcA C1B regulatory domain. The <i>pkcA</i>^G579R mutation has a reduced activation of the downstream <u>M</u>itogen-<u>A</u>ctivated <u>P</u>rotein <u>K</u>inase, MpkA, resulting in the altered expression of genes encoding cell wall-related proteins, markers of endoplasmic reticulum stress and the unfolded protein response. Furthermore, PkcA^G579R is involved in the formation of proper conidial architecture and protection to oxidative damage. The <i>pkcA</i>^G579R mutant elicits increased production of TNF-α and phagocytosis but it has no impact on virulence in a murine model of invasive pulmonary aspergillosis. These results highlight the importance of PkcA to the CWI pathway but also indicated that additional regulatory circuits may be involved in the biosynthesis and/or reinforcement of the <i>A</i>. <i>fumigatus</i> cell wall during infection.</p></div