PMID- 17947229
OWN - NLM
STAT- MEDLINE
DCOM- 20080320
LR  - 20210209
IS  - 0021-9258 (Print)
IS  - 0021-9258 (Linking)
VI  - 283
IP  - 2
DP  - 2008 Jan 11
TI  - Characterization of the N-acetyl-5-neuraminic acid-binding site of the 
      extracytoplasmic solute receptor (SiaP) of nontypeable Haemophilus influenzae 
      strain 2019.
PG  - 855-65
AB  - Nontypeable Haemophilus influenzae is an opportunistic human pathogen causing 
      otitis media in children and chronic bronchitis and pneumonia in patients with 
      chronic obstructive pulmonary disease. The outer membrane of nontypeable H. 
      influenzae is dominated by lipooligosaccharides (LOS), many of which incorporate 
      sialic acid as a terminal nonreducing sugar. Sialic acid has been demonstrated to 
      be an important factor in the survival of the bacteria within the host 
      environment. H. influenzae is incapable of synthesizing sialic acid and is 
      dependent on scavenging free sialic acid from the host environment. To achieve 
      this, H. influenzae utilizes a tripartite ATP-independent periplasmic 
      transporter. In this study, we characterize the binding site of the 
      extracytoplasmic solute receptor (SiaP) from nontypeable H. influenzae strain 
      2019. A crystal structure of N-acetyl-5-neuraminic acid (Neu5Ac)-bound SiaP was 
      determined to 1.4A resolution. Thermodynamic characterization of Neu5Ac binding 
      shows this interaction is enthalpically driven with a substantial unfavorable 
      contribution from entropy. This is expected because the binding of SiaP to Neu5Ac 
      is mediated by numerous hydrogen bonds and has several buried water molecules. 
      Point mutations targeting specific amino acids were introduced in the putative 
      binding site. Complementation with the mutated siaP constructs resulted either in 
      full, partial, or no complementation, depending on the role of specific residues. 
      Mass spectrometry analysis of the O-deacylated LOS of the R127K point mutation 
      confirmed the observation of reduced incorporation of Neu5Ac into the LOS. The 
      decreased ability of H. influenzae to import sialic acid had negative effects on 
      resistance to complement-mediated killing and viability of biofilms in vitro, 
      confirming the importance of sialic acid transport to the bacterium.
FAU - Johnston, Jason W
AU  - Johnston JW
AD  - Department of Microbiology, University of Iowa, Iowa City, Iowa 52242, USA.
FAU - Coussens, Nathan P
AU  - Coussens NP
FAU - Allen, Simon
AU  - Allen S
FAU - Houtman, Jon C D
AU  - Houtman JC
FAU - Turner, Keith H
AU  - Turner KH
FAU - Zaleski, Anthony
AU  - Zaleski A
FAU - Ramaswamy, S
AU  - Ramaswamy S
FAU - Gibson, Bradford W
AU  - Gibson BW
FAU - Apicella, Michael A
AU  - Apicella MA
LA  - eng
SI  - PDB/3B50
GR  - AI024616/AI/NIAID NIH HHS/United States
GR  - AI07260-21/AI/NIAID NIH HHS/United States
GR  - AI30040/AI/NIAID NIH HHS/United States
PT  - Journal Article
PT  - Research Support, N.I.H., Extramural
DEP - 20071018
PL  - United States
TA  - J Biol Chem
JT  - The Journal of biological chemistry
JID - 2985121R
RN  - 0 (Bacterial Proteins)
RN  - 0 (Lipopolysaccharides)
RN  - 0 (Receptors, Cell Surface)
RN  - 0 (Sialic Acids)
RN  - 0 (lipid-linked oligosaccharides)
RN  - 0 (sialic acid receptor)
RN  - GZP2782OP0 (N-Acetylneuraminic Acid)
SB  - IM
MH  - Bacterial Proteins/chemistry/metabolism
MH  - Binding Sites
MH  - Cell Membrane/*metabolism
MH  - Cloning, Molecular
MH  - Cytoplasm/metabolism
MH  - Escherichia coli/drug effects/genetics/metabolism
MH  - Genotype
MH  - Haemophilus influenzae/drug effects/genetics/*metabolism
MH  - Lipopolysaccharides/pharmacology
MH  - Models, Molecular
MH  - Mutagenesis, Site-Directed
MH  - N-Acetylneuraminic Acid/chemistry/metabolism
MH  - Phenotype
MH  - Point Mutation
MH  - Protein Conformation
MH  - Receptors, Cell Surface/genetics/*metabolism
MH  - Sialic Acids/*metabolism
MH  - Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
EDAT- 2007/10/20 09:00
MHDA- 2008/03/21 09:00
CRDT- 2007/10/20 09:00
PHST- 2007/10/20 09:00 [pubmed]
PHST- 2008/03/21 09:00 [medline]
PHST- 2007/10/20 09:00 [entrez]
AID - S0021-9258(20)68996-7 [pii]
AID - 10.1074/jbc.M706603200 [doi]
PST - ppublish
SO  - J Biol Chem. 2008 Jan 11;283(2):855-65. doi: 10.1074/jbc.M706603200. Epub 2007 
      Oct 18.